Edward Franklin

Seamus Browne

Jerrard Hayes

Coilin Boland

Aisling Dunne

Gordon Elliot

Timothy J Mantle

Activation of biliverdin-IXalpha reductase by inorganic phosphate and related anions

Keywords: Biliverdin reductase, pH optima, anion activation, Xenopus tropicalis, phosphate, nicotinamide adenine dinucleotide

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Introduction

The haem catabolic pathway is currently receiving attention in a number of fields with the observation that induction of haem oxygenase-1 (HO-1) is cytoprotective [1; 2]. Attempts to relate this to production of the immediate product biliverdin [3; 4] and/or subsequent reduction to bilirubin [START_REF] Kapitulnik | Bilirubin: an endogenous product of heme degradation with both cytotoxic and cytoprotective properties[END_REF] or to a redox cycling mechanism between these two linear tetrapyrroles [START_REF] Baranano | Biliverdin reductase: a major physiologic cytoprotectant[END_REF] are unresolved, however there is clear evidence that biliverdin has some unexpected and dramatic effects. Bach and coworkers have presented intriguing evidence that administration of biliverdin is cytoprotective for heart, colon and liver in animal transplantation studies [7; 8; 9]. In a striking report Bach and co-workers have shown that short term treatment (3 weeks) with biliverdin is sufficient to induce tolerance in a recipient to the donor heart for 120 days [START_REF] Yamashita | Biliverdin, a natural product of heme catabolism, induces tolerance to cardiac allografts[END_REF]. The introduction of an allogeneic third heart at 120 days was rejected whereas the introduction of a third syngeneic heart was accepted, clearly indicating tolerance.

Despite the current interest in the metabolism of linear tetrapyrroles, biliverdin-IXα reductase (BVR-A) has received relatively little attention. The reaction catalysed by BVR-A is illustrated in Scheme 1. BVR-A is a cytosolic monomer (34kDa), although the human enzyme migrates anomalously on SDS/PAGE with a mobility corresponding to a molecular weight reported variously as 46kDa [START_REF] Rigney | Some physical and immunological properties of ox kidney biliverdin reductase[END_REF] or 41kDa [START_REF] Maines | Purification and characterization of human biliverdin reductase[END_REF]. BVR-A catalyses the transfer of the B-face hydrogen [START_REF] Ennis | Cloning and over-expression of rat kidney biliverdin IX alpha reductase as a fusion protein with glutathione Stransferase: stereochemistry of NADH oxidation and evidence that the presence of the glutathione S-transferase domain does not effect BVR-A activity[END_REF] to a variety of verdin acceptors [START_REF] Cunningham | Studies on the specificity of the tetrapyrrole substrate for human biliverdin-IXalpha reductase and biliverdin-IXbeta reductase. Structure-activity relationships define models for both active sites[END_REF]. The structure of the rat enzyme [START_REF] Kikuchi | Crystal structure of rat biliverdin reductase[END_REF] shows that the enzyme contains two domains, an N-terminal domain characteristic of a dinucleotide binding fold (Rossmann fold) and a C-terminal domain that is predominantly an antiparallel six-stranded beta-sheet. A structure for the rat enzyme with NADH bound has also been reported [START_REF] Whitby | Crystal structure of a biliverdin IXalpha reductase enzyme-cofactor complex[END_REF], however the adenine moiety folds back over the nicotinamide ring and this may, as the authors discuss, be an artefact of crystallisation where lysine-209 from a symmetryrelated molecule precludes the adenine ring adopting the open structure predicted to exist in solution [START_REF] Kikuchi | Crystal structure of rat biliverdin reductase[END_REF].

BVR-A is a small enzyme that exhibits complicated kinetic behaviour. The enzyme shows pronounced substrate inhibition (which may be modulated by intracellular biliverdin-binding proteins [START_REF] Phillips | On the possible role of biliverdin stimulation of cyclic AMP levels as a trigger for liver regeneration in the rat[END_REF]) and also has two distinct pH optima depending on whether NADH or NADPH is the pyridine nucleotide. NADH is preferentially used between pH 6 and 7 while NADPH is the preferred cofactor between pH 8.5 to 8.7. This behaviour is seen with both native and recombinant human and rat BVR-A [17; 18; 19; 20], the ox kidney [START_REF] Rigney | The kinetics of ox kidney biliverdin reductase in the pre-steady state. Evidence that the dissociation of bilirubin is the rate-determining step[END_REF] and the pig spleen enzymes [START_REF] Noguchi | Purification and properties of biliverdin reductases from pig spleen and rat liver[END_REF]. Interestingly one notable exception to this dual pH behaviour is the cyanobacterial BVR-A from Synechocystis PCC6803 which displays maximal activity at pH 5.8 when using either NADPH or NADH as cofactor [START_REF] Schluchter | Characterization of cyanobacterial biliverdin reductase. Conversion of biliverdin to bilirubin is important for normal phycobiliprotein biosynthesis[END_REF]. During a re-investigation of this phenomenon (extended to include the recombinant enzymes from Xenopus tropicalis and mouse) we have discovered that the enzyme activity with NADH as cofactor is markedly activated by inorganic phosphate at neutral pH and present a novel mechanism to explain this observation.

Materials and Methods

Details on the purification of the salmon [START_REF] Elliott | Purification and properties of salmon liver biliverdin reductase[END_REF], rat [START_REF] Ennis | Cloning and over-expression of rat kidney biliverdin IX alpha reductase as a fusion protein with glutathione Stransferase: stereochemistry of NADH oxidation and evidence that the presence of the glutathione S-transferase domain does not effect BVR-A activity[END_REF], human [START_REF] Cunningham | Studies on the specificity of the tetrapyrrole substrate for human biliverdin-IXalpha reductase and biliverdin-IXbeta reductase. Structure-activity relationships define models for both active sites[END_REF] and Synechocystis [START_REF] Schluchter | Characterization of cyanobacterial biliverdin reductase. Conversion of biliverdin to bilirubin is important for normal phycobiliprotein biosynthesis[END_REF] by affinity chromatography on glutathione-agarose (Chromatrin). Site directed mutagenesis of the rat enzyme was carried out using PCR as described by [START_REF] Fisher | Modification of a PCR-based site-directed mutagenesis method[END_REF].

Initial rate kinetics were conducted by following the production of bilirubin by monitoring the increase in absorbance at 460 nm as described previously [START_REF] Cunningham | Studies on the specificity of the tetrapyrrole substrate for human biliverdin-IXalpha reductase and biliverdin-IXbeta reductase. Structure-activity relationships define models for both active sites[END_REF].

Assays were conducted at The stopped flow experiments were conducted using an Applied Photophysics spectrometer and Pro-K global analysis software.

The following buffers (100mM) were used to determine the effect of pH on the activity of BVR-A: sodium acetate (pH 5 -6), sodium citrate (pH 5 -6.5), sodium phosphate (pH 6.5 -8) and Tris-HCl (pH 8 -9). Assays for BVR-A activity were generally conducted in the absence of BSA. The addition of BSA gives an enhanced ∆A 460 and maintains linear initial rates for longer, however it introduces complications when interpreting kinetic results since the total concentration of biliverdin is not identical with the free concentration as a significant fraction binds to BSA (or any other biliverdin binding protein [26; 16]). Biliverdin-IXα was prepared by oxidation of bilirubin-IXα [START_REF] Mcdonagh | The Porphyrins[END_REF].

Results and Discussion

The effects of pH on the stability of various BVR-As.

To determine the stability of the rat, murine and human recombinant BVR-A, the enzymes were pre-incubated in various buffers over the pH range 5 -9. The human enzyme (Fig. 1a) lost no more than 5 -10% of its activity even at the extremes of pH tested (pH5 and pH9). Under the conditions examined the enzyme was relatively dilute (0.1 mg/ml) and was pre-incubated for 10 minutes at the various pH values. In kinetic studies the reaction was generally initiated by the addition of enzyme and the initial rate measured from the initial 10 -20 seconds of the reaction. It is unlikely therefore that any significant inactivation of the human enzyme occurred in these initial rate experiments. The rat enzyme exhibited a similar tolerance to pH but was marginally less stable at alkaline values, losing 25% of its activity at pH 9 after a 10 minute pre-incubation (Fig. 1a).

The mouse enzyme is considerably less stable at both acid and alkaline pH values and even at pH values between 6 and 8 loses 10 -20% activity after 10 minutes incubation at 30 o C (Fig. 1a). The relative instability of murine BVR-A at acid pH values prompted a further investigation to determine the kinetics of inactivation at pH 4 and 4.5. After 10 minutes pre-incubation at pH 4.5, only 10%

of the activity remained (Fig. 1b). However 50% activity remains after 2 minutes pre-incubation at pH 4.5 (Fig. 1b) and extrapolation suggests that measurements up to 30 seconds at pH 4.5 will yield 90% of the original activity. It is not possible to extend this level of confidence to pH 4 where only 50% activity remains after 30 seconds pre-incubation (Fig. 1b). The degradation of NADH and NADPH under acid conditions has been reported in numerous studies see e.g. [START_REF] Wu | Stability of NADPH: effect of various factors on the kinetics of degradation[END_REF]. It is clear from these studies that the pH has a significant effect on the rate of NADPH and NADH degradation. At 30°C, the half life of NADPH was reported as just 38 minutes at pH 5 [START_REF] Wu | Stability of NADPH: effect of various factors on the kinetics of degradation[END_REF]. As a result stock solutions of NAD(P)H in water were prepared fresh each day and stored on ice.

The effect of pH on the activity of BVR-A

It is important to note that of all the BVR-A enzymes characterised to date the Synechocystis PCC6803 and salmon enzymes are the only forms where activity has been surveyed over the entire pH range studied using both nucleotides [23; 29] and these, together with the eel (Anguilla japonica) enzyme [START_REF] Fang | Characterisation and purification of biliverdin reductase from the liver of eel, 'Anguilla japonica[END_REF], support significant activity at acid pH values.

A series of reactions were set up to determine the effects of pH on the activity of human, rat and Xenopus BVR-A and the results are presented in Figs 2a,b andc.

During the course of these experiments two interesting observations were made.

The first is that the use of phosphate buffer substantially increased the rate for the NADH-dependent reaction over the pH range (from pH 6-8) used in these experiments for the human and rat enzymes. The second point worth noting is that the NADPH-dependent activity of the human and rat enzymes is clearly not limited to the pH range of 7.5 to 9.

The activity of the Xenopus enzyme exhibited a pH optima of 4.75, which is similar to the behaviour of the Synechocystis enzyme [START_REF] Schluchter | Characterization of cyanobacterial biliverdin reductase. Conversion of biliverdin to bilirubin is important for normal phycobiliprotein biosynthesis[END_REF]. The Xenopus enzyme is similar to the salmon enzyme [29] in that both are more active with NADH than NADPH.

The effects of various anions on the activity of human BVR-A

Fig. 3 shows the effect of increasing concentrations of phosphate buffer (pH 7)

on the activity of human BVR-A with NADH as the cofactor. The data fits well to the Hill equation, as illustrated, however there are probably effects due to ionic strength in addition to the specific activation by phosphate anion. The activity in 5 mM phosphate is 0.04 µmol.min -1 .mg -1 and in 200mM phosphate this increases 37.5 fold to 1.5 µmol.min -1 .mg -1 . To determine whether this effect was specific to phosphate a range of concentrations of various buffers at pH 7 was tested.

Assays were performed with 20 µM biliverdin and 700 µM NADH at 30°C in various concentrations of sodium phosphate (2, 5, 10, 25, 50, 75, 100 and 200 mM) and sodium carbonate, Tris-HCl and sodium citrate (all at 10, 50, 100 and 200 mM). The reaction was started by the addition of enzyme. The pH of each assay were tested before and after the assay to ensure that pH did not alter through the course of the assay especially when using salts at the limits of their buffering capacity.

The results of the experiment detailed in Fig. 3 clearly demonstrate that phosphate is a powerful activator when NADH is used as cofactor. There is a modest effect of ionic strength as shown by the increase in initial rates seen with increasing concentrations of sodium citrate, Tris-HCl and sodium carbonate up to the 200 mM. However the effect of phosphate on enzyme activity is particularly marked. Interestingly pyrophosphate also displayed an activatory effect, indeed the potency observed was greater than that obtained with phosphate (Fig. 4). It is also clear from Fig. 4 that the marked activation seen with both phosphate and pyrophosphate is specific for NADH. There is only a very modest increase in activity seen with NADPH as cofactor over the same range. The basis for the activation however was unclear from these results. Phosphate may allow substrates to bind more effectively to the enzyme and/or enhance turnover.

The effects of phosphate on the NADH dependent activity of human BVR-A

A series of saturation curves were generated with NADH as the variable substrate (20 µM to 3.2 mM) in 10, 50, 100, and 200mM sodium phosphate pH 7.

Biliverdin-IXα at a final concentration of 20 µM was used as substrate and the reaction was performed at 30°C. Data points were fitted to the Michaelis equation and the rectangular hyperbolas generated by these fits were used to obtain the apparent K m and V max (Table 1). Increasing phosphate from 10mM to 200mM resulted in a dramatic decrease in the apparent Km for NADH (150-fold) with no significant change in the apparent Vmax. Clearly inorganic phosphate promotes NADH binding without enhancing turnover.

The effect of phosphate on the burst rate constant of human BVR-A.

Stopped-flow studies on ox kidney [START_REF] Rigney | The kinetics of ox kidney biliverdin reductase in the pre-steady state. Evidence that the dissociation of bilirubin is the rate-determining step[END_REF] and rat [START_REF] Ennis | Cloning and over-expression of rat kidney biliverdin IX alpha reductase as a fusion protein with glutathione Stransferase: stereochemistry of NADH oxidation and evidence that the presence of the glutathione S-transferase domain does not effect BVR-A activity[END_REF] BVR-A shows a burst reaction which has been attributed to the slow release of bilirubin from an enzyme.NADP.bilirubin complex. The effect of phosphate on the pre-steady state kinetics was determined by monitoring the increase in absorbance at 460nm at The effect of pH on the activation by phosphate of human BVR-A using NADH as cofactor.

The effect of pH on the activity of human BVR-A with increasing concentrations of phosphate was also investigated (Fig. 6). The activity was measured in 100 mM sodium citrate over the pH range 5-7 with 7 µm biliverdin-IXα and 100 µM NADH as substrates. The concentrations of phosphate employed were 5, 10, 15, 20, 40, 60, 80, 100 mM. It is clear that at pH 5 the activation by phosphate anion is less potent than that seen at more neutral pH values. By plotting A 50 (the concentration of activator giving 50% activation) against pH an apparent pK of approximately 4.9 can be identified (insert to Fig. 6). Deprotonation of this residue increases the affinity for phosphate anion 6-fold. A possible candidate for this pK can be tentatively identified as glutamate-75. The side chain of glu75

interacts with arg44 (one of the residues that interacts with inorganic phosphate in the rat structure) and is predicted to do so more strongly as the carboxylate anion. A weaker interaction as the protonated carboxyl may allow arg44 to adopt an alternative conformation that partially destabilises the arg44 interaction with phosphate.

Studies on phosphate activation of rat BVR-A: identification of a putative 2'-phosphate binding site

In an effort to further understand the nature of the activation of the NADHdependent activity of human BVR-A by phosphate and related anions it was decided to analyse the two BVR-A structures then available ([14] [START_REF] Whitby | Crystal structure of a biliverdin IXalpha reductase enzyme-cofactor complex[END_REF]) for the rat enzyme. Although not commented on by the authors, rat BVR-A [START_REF] Whitby | Crystal structure of a biliverdin IXalpha reductase enzyme-cofactor complex[END_REF] [15] co-crystallised with three inorganic phosphate molecules [START_REF] Whitby | Crystal structure of a biliverdin IXalpha reductase enzyme-cofactor complex[END_REF].

Interestingly, one of these phosphates occupies a site which has been proposed as the binding pocket for the 2'-phospho ester of NADPH [START_REF] Kikuchi | Crystal structure of rat biliverdin reductase[END_REF]. In light of this information it was decided to look at the residues involved in binding phosphate in more detail. As shown in Fig. 7, serine-43, arginine-44 and arginine-45 form an intricate lattice of ionic interactions and hydrogen bonds with inorganic phosphate.

Effect of phosphate concentration on the activity of wild type and "2'phosphate pocket" mutants of rat BVR-A Fig. 8 shows the effect of phosphate concentration on the activity of the rat enzyme (wild type) in Tris-HCl buffer. As with the human enzyme there is a marked activation by phosphate with an A 50 value of 20 mM. Although all three of the desired mutants were created only the S43A and the R44A mutants were sufficiently stable to examine kinetically. The A 50 for the S43 mutant increases significantly (Fig. 8) to 60 mM indicating that significant binding energy results from the interaction between serine 43 and the phosphate anion. A more dramatic result was seen with the R44A mutant which is not activated significantly by increasing concentrations of phosphate when NADH is used as cofactor (Fig. 8). Mutation of these two residues in the "2'-phosphate pocket", decreases (S43A), and abolishes (R44A) the ability of inorganic phosphate to activate the NADH-dependent reduction of biliverdin, clearly implicating this as the site involved in the phosphate-activation phenomenon. We have also confirmed an earlier report [START_REF] Kikuchi | Crystal structure of rat biliverdin reductase[END_REF] that the R44A mutation equalises the apparent Km for NADH and NADPH (data not shown) again implicating this as the binding site for the 2'-phosphoester of NADPH.

The conclusion from this study that NAD(P)H binds in an extended conformation and that inorganic phosphate binds to the 2'-phosphate pocket for NAD(P)H has recently been confirmed in the crystal structure of human BVR-A with NADPH which has been deposited in the Protein Data Bank by the Structural Genomics Consortium with code 2H63. Our model for the mechanism of activation of the NADH-dependent activity of BVR-A by inorganic phosphate therefore proposes that inorganic phosphate mimicks the 2'-phosphoester of NADPH. There are clearly questions remaining concerning the nature of this complex as an additional oxygen atom is present in the enzyme.NADH.Pi complex when compared with the enzyme.NADPH complex. Indeed the ability of pyrophosphate to activate suggests that at least some reorganisation of the enzyme structure is necessary to achieve the ternary complexes with anions and this will be the focus of further studies. The hypothesis that NADH binding can be augmented by inorganic phosphate "mimicking" the 2'-phosphate of NADPH is not entirely novel as a similar conclusion has been reached by [START_REF] Christensen | Magnesium and phosphate ions enable NAD binding to methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase[END_REF] [START_REF] Christensen | Magnesium and phosphate ions enable NAD binding to methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase[END_REF] for the Mgdependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC).

These authors point out that NMDMC uses phosphate and magnesium to adapt an NADP site for NAD binding.

The effect of pH and biliverdin-IXα concentrations on the initial rate using NADPH as cofactor.

At 2 µM biliverdin-IXα the human enzyme is active over the entire pH range studied (Fig. 9). As the substrate concentration is increased to 5 and the initial rate is significantly suppressed at neutral pH (due to pronounced substrate inhibition) which is far less severe under both acid and alkaline conditions (Fig. 9). This results in a shift in the apparent pH optimum from 8 to 8.5, depending on the biliverdin concentration used in the assay. The increased potency of substrate inhibition at neutral pH was also clearly evident in the time course of the reaction. An increase in rate during the time course of the reaction was observed as the substrate concentration decreased below substrate inhibitory levels. This was particularly apparent at neutral pH values, where substrate inhibition is pronounced, while at the acid and alkaline limbs, where substrate inhibition was not apparent more normal P(t) curves were observed (data not shown). It is still not clear why NADPH supports a significant rate at pH 8.5 to 9 but that NADH in unable to achieve this.

It should be noted that the approximate concentrations of pyridine nucleotides in rat liver have been reported as 120 µM for NADPH, 35 µM for NADP, 70 µM for NADH, and 500 µM for NAD [32; 33]. With the apparent K m for NADPH of biliverdin reductases being in the low micro molar range, typically 1-10µM, the enzyme is most likely saturated with NADPH in vivo. In contrast, the apparent K m for NADH is generally reported between 300 and 500µM, and with cytosolic concentrations of NADH generally around 70 µM this is unlikely to be a physiologically significant substrate. In well oxygenated perfused rat liver the cytosolic concentration of inorganic phosphate has been reported as 5.3mM [START_REF] Desmoulin | Published on 3 Apr 2007 as manuscript BJ20061651 © 2007 Biochemical Society intracellular pH and phosphorylation state during normoxia, hypoxia and ethanol perfusion, in the perfused rat liver[END_REF] Biochemical Journal Immediate Publication. Published on 3 Apr 2007 as manuscript BJ20061651

© 2007 Biochemical Society and therefore is not predicted to impinge on NADH-dependent activity. However inorganic phosphate in mitochondria can rise to 25mM and as this subcellular location has a higher NADH/NAD ratio and has recently been reported to contain BVR-A [START_REF] Converso | HO-1 is located in liver mitochondria and modulates mitochondrial heme content and metabolism[END_REF], inorganic phosphate may promote NADH-dependent BVR-A activity in mitochondria. The enzyme (5 µg) was assayed using 700 µM NADH as cofactor and 10 µM biliverdin as substrate at pH 7 in ( ) sodium phosphate, and in ( ) sodium pyrophosphate. Also shown are the results when hBVR-A was assayed using 100 µM NADPH and 10 µM biliverdin as substrate in sodium phosphate ( ) pH 7 at the concentrations indicated.

Legends to

Table 1

The apparent K M and V max of human BVR-A for NADH with increasing concentrations of sodium phosphate.

Data points in Fig. 6 were fitted to the Michaelis equation and the rectangular hyperbolas generated by these fits were used to obtain the apparent K m and V max .

  enzymes have been reported previously. The pBS152V Synechocystis BVR-A encoding plasmid was a kind gift from Dr. W. Schluchter, of the Department of Molecular and Cell Biology, University of California, Berkeley, California. The mouse enzyme was cloned by RT/PCR using mRNA from Balb/c mouse liver. The specific primer used for the reverse transcriptase reaction was TGAAGCTTCCTCCTCACTGCTTCCGG and the primers for the PCR reaction were, forward primer: CCTCTAGAATGAGTACTGAGCCAA and reverse primer: CCAAGCTTCCTCCTCACTGCTTCC. The PCR product was digested with XbaI and HindIII to allow directional cloning into the maltose-binding protein fusion vector ppMAL. DNA sequencing of the murine BVR-A cDNA construct was carried out and compared with four EST sequences. The cDNA sequence of EST-1 (Accession Number AK002231.1) appears to have a stop codon 21 base pairs after the stop codon of all other cDNA sequences aligned including that of the cloned product. An additional variation occurs in the codon for leucine-27 (TTG). In two of these sequences (accession numbers AK002231.1 and AK010847.1) and also the murine ppMAL-mBVR-A construct, the codon is as expected, however in 2 other EST's (accession numbers BC052146.1 and NM_026678.3), codon-27 reads (TCG) which codes for lysine at this position. The MBP-mBVR-A fusion protein was produced in E-coli strain BL21 transformed with ppMAL-mBVR-A. After induction with IPTG (0.2mM for 12 hours at 27 o C) the cytosol was loaded onto an amylose affinity column (1.5 x 10cm) to bind the MBP/BVR-A fusion protein. This was washed extensively with 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7 and the fusion protein was then eluted with 10 mM maltose in 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7. The eluate was tested for BVR-A activity and the peak fractions pooled. From 1 litre of culture 20 mg MBP fusion was routinely obtained. The MBP was cleaved using PreScission™ Protease (Amersham Biosciences). The enzyme from Xenopus tropicalis was cloned from the I.M.A.G.E cDNA clone (IMAGE:5308696) which was acquired from UK HGMP-RC, Hinxton, primers an amplified product of the predicted size (~882 base pairs) was obtained and cut with BamHI and XhoI to allow directional cloning into pGEX-KG. The resulting plasmid (pGEX-KG-XeBVR-A) was transformed into E.coli BL21 and the GST-fusion protein isolated

  23°C. Human BVR-A (1.6 µM) in 100mM sodium citrate pH 7.2 was rapidly mixed with 1.4 mM NADH, 14 µM biliverdin-IXα in varying concentrations of sodium phosphate pH 7.2 (Fig.5). The concentration of phosphate in the substrate syringe was varied to give final concentrations of 5, 10, 20, 50, 100 and 200 mM.The results of these stopped flow experiments were fitted to a curve of single exponential with slope. The steady state rate showed that at 23 o C there is a pronounced activation by phosphate anion (8-fold compared with the rate in Tris-Cl) and the burst rate constants (k b ) obtained from these experiments are shown in the inset to Fig.5. There is a modest effect of phosphate concentration on the burst rate constant which essentially doubles between 0 and 50 mM phosphate from 0.15 s -1 to 0.28s -1 .

  Scheme 1 BVR-A catalysed reduction of biliverdin-IX

Figure 2

 2 Figure 2 The effect of pH on the activity of BVR-A The results are shown for the (a) human, (b) rat and (c) Xenopus enzymes. The enzyme was assayed at the pH indicated in the following buffers sodium citrate ), sodium phosphate acetate and Tris/HCl using 700 µM NADH as cofactor and sodium citrate phosphate sodium acetate and Tris/HCl using 100 µM NADPH as cofactor. A final concentration of 10 µM biliverdin was used.

Figure 3 (

 3 Figure 3 (a) The effect of various buffers on the NADH-dependent activity of human BVR-AThe activity of the human enzyme was assayed in the presence of 700µM NADH and 20 µM biliverdin as substrate at 30°C in the various buffers at the concentrations indicated. The buffers used were sodium phosphate sodium carbonate Tris/HCl and sodium citrate at pH 7.

Figure 4

 4 Figure 4 The effect of various anions on the NADH-and NADPH-dependent activity of human BVR-A.

Figure 5

 5 Figure 5 The effect of phosphate on the pre-steady burst of hBVR-AThe activity of the human enzyme (0.8 µM) was assayed in the presence of 700 µM NADH and 7 µM biliverdin with increasing concentrations of phosphate (0, 5, 10, 50, 100 and 200 mM) in 50 mM Tris pH 7.2.

Figure 6

 6 Figure 6 Effect of pH on the phosphate-dependent activation of hBVR-AThe activity of human BVR-A was measured in 100mM sodium citrate over the pH range 5-7 ( pH 5, pH 5.5, pH 6, pH 6.5, and pH 7) with 7 µm biliverdin-IXα and 700 µM NADH. Phosphate concentration was increased from 0 to 100mM phosphate at each pH measured. Inset is a plot of A 50 against pH fitted to a single pK.

Figure 7

 7 Figure 7 The inorganic phosphate pocket of rBVR-AThe coordinates used for this Figure are taken from the crystal structure of the rat enzyme reported by[START_REF] Whitby | Crystal structure of a biliverdin IXalpha reductase enzyme-cofactor complex[END_REF] [START_REF] Whitby | Crystal structure of a biliverdin IXalpha reductase enzyme-cofactor complex[END_REF]. The structure was downloaded from the Protein Data Bank[START_REF] Berman | The Protein Data Bank[END_REF], and graphics generated using the pyMol program published by DeLano Scientific[START_REF] Delano | The PyMOL Molecular Graphics System[END_REF]. Red dots represent water molecules. A surface diagram of the residues is shown in (a). The phosphate pocket is clearly defined with a large distribution of positive charges contributed by R 44 and R45 which lie along the walls of this pocket (coloured blue) and are seen to coordinate with the phosphate anion of the crystal structure (shown in b for clarity).

Figure 7 c

 7 Figure 7 c highlights the salt bridge formation between R44 and E75. This interaction may contribute to the stabilisation of the loop between B-sheets and and the αD helix in the rat crystal structure. Residues S43, R44 and R45

Figure 8

 8 Figure 8 The effect of phosphate on the activity of the wild type, S43A and R44A mutant rBVR-A The activity of wild type rat BVR-A R45A BVR-A and S44A BVR-A was measured in 50 mM Tris/HCl at pH 7.2 with 700 µM NADH and 10 µm biliverdin-IXα as substrate over the phosphate concentrations range (0 to 250 mM).

Figure 9

 9 Figure 9 The effect of biliverdin concentration on the pH dependence of hBVRA activity. The effect of biliverdin concentration on the pH dependence of hBVRA activity was investigated. The reaction was monitored at 460 nm at 30°C, and contained a final concentration of 100µM NADPH with 2, 5 and 20µM biliverdin-IXα over the pH range 4.5-8.5.

  Figure 1

Figure 3 Biochemical

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Figure 4 Biochemical

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Figure 5 Biochemical

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Figure 6 Biochemical

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Figure 8 Biochemical

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Figure 9 Biochemical

 9 Figure 9

Biochemical Journal Immediate Publication. Published on 3 Apr 2007 as manuscript BJ20061651 © 2007 Biochemical Society

	enzyme. To determine the stability of BVR-A over the pH range studied the
	various enzymes were diluted 10 fold to a concentration of 0.1 mg/ml in 100 mM
	buffer at each pH. The diluted mixtures were incubated at 30°C for 10 minutes in
	the buffers indicated, after which time 10µl aliquots were assayed in 100mM Tris-
	HCl pH 8 (final volume 2ml), with a final concentration of 100 µM NADPH and
	10µM biliverdin. Initial rates are described as µmoles or nmoles produced per
	minute per mg enzyme. For some experiments, particularly the stopped-flow
	experiments, the rates are expressed as change in absorbance ( A) per minute.
	30 o C using a Unicam 8625 UV/Vis spectrophotometer
	connected to a Kipp and Zonen flatbed chart recorder. For routine assays the
	incubation mixture contained 0.1 M sodium phosphate buffer pH 7.2, 700 µM
	NADH, 10 µM biliverdin-IXα and the reaction was started by the addition of

Biochemical Journal Immediate Publication. Published on 3 Apr 2007 as manuscript BJ20061651 © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 3 Apr 2007 as manuscript BJ20061651 © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 3 Apr 2007 as manuscript BJ20061651 © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 3 Apr 2007 as manuscript BJ20061651 © 2007 Biochemical Society

Table 1 Biochemical Journal Immediate Publication. Published on 3 Apr 2007 as manuscript BJ20061651

 1

	[Phosphate] (mM)	appKm NADH (mM)	appVmax (µmole.min -1 .mg -1 )
	10	39.20 +/-69	2.00 +/-3.77
	50	3.05 +/-0.74	2.03 +/-0.29
	100	0.79 +/-0.18	2.00 +/-0.21
	200	0.22 +/-0.02	2.12 +/-0.07

Biochemical Journal Immediate Publication. Published on 3 Apr 2007 as manuscript BJ20061651 © 2007 Biochemical Society

© 2007 Biochemical Society

Acknowledgements:

This work was supported by a grant from Science Foundation Ireland.

Biochemical Journal Immediate Publication. Published on 3 Apr 2007 as manuscript BJ20061651


Francesca Malerba

Andrea Bellelli

Alessandra Giorgi

Francesco Bossa

Roberto Contestabile
email: roberto.contestabile@uniroma1.it

The mechanism of addition of pyridoxal 5'-phosphate to Escherichia coli apo-serine hydroxymethyltransferase

Keywords: Pyridoxal phosphate, serine hydroxymethyltransferase, cofactor PLP, pyridoxal 5'-phosphate; PNP, pyridoxine 5

Previous studies suggest that the addition of pyridoxal 5'-phosphate to apo-serine hydroxymethyltransferase from Escherichia coli is the last event in the enzyme's folding process. We propose a mechanism for this reaction based on quenched-flow, stopped-flow and rapid scanning stopped-flow experiments. All experiments were performed with an excess of apo-enzyme over cofactor, since excess pyridoxal phosphate results in a second molecule of cofactor binding to Lys346, which is part of the tetrahydropteroylglutamate binding site. The equilibrium between the aldehyde and hydrate forms of the cofactor affects the kinetics of addition to the active site. Direct evidence of the formation of an intermediate aldimine between the cofactor and the active site lysine was obtained. The results have been interpreted according to a three-step mechanism in which: 1) both aldehyde and hydrate forms of the cofactor bind rapidly and non-covalently to the apo-enzyme; 2) only the aldehyde form reacts with the active site lysine to give an intermediate internal aldimine with unusual spectral properties; 3) a final conformational change gives the native holo-enzyme.

INTRODUCTION

The encounter between cofactors and apo-proteins in the cell is a crucial event that can occur at different stages of polypeptide folding and affect it to a variable extent [START_REF] Higgins | How do cofactors modulate protein folding?[END_REF][START_REF] Wittung-Stafshede | Role of cofactors in protein folding[END_REF]. The form in which the cofactor is available in the intracellular medium may also differ according to its reactivity or toxicity [START_REF] Wittung-Stafshede | Role of cofactors in folding of the blue-copper protein azurin[END_REF]. The role of pyridoxal 5'-phosphate (PLP) in folding and structural stabilisation apparently varies among the enzymes which use it as cofactor [START_REF] Bettati | Role of pyridoxal 5'-phosphate in the structural stabilization of O-acetylserine sulfhydrylase[END_REF][START_REF] Groha | Refolding and reactivation of Escherichia coli tryptophan synthase beta2 subunit after inactivation and dissociation in guanidine hydrochloride at acidic pH[END_REF][START_REF] Herold | Coenzyme binding of a folding intermediate of aspartate aminotransferase detected by HPLC fluorescence measurements[END_REF][START_REF] Bertoldi | Folding pathway of the pyridoxal 5'-phosphate C-S lyase MalY from Escherichia coli[END_REF][START_REF] Cai | The affinity of pyridoxal 5'-phosphate for folding intermediates of Escherichia coli serine hydroxymethyltransferase[END_REF]. This is not surprising, considering that there are at least five evolutionary unrelated families of such enzymes, corresponding to completely different protein folds (fold-types I-V) [START_REF] Grishin | Modeling of the spatial structure of eukaryotic ornithine decarboxylases[END_REF].

The mechanism of PLP homeostasis and targeting to the newly synthesised apoenzymes is largely unknown. A first step in elucidating this process is to understand how PLP reacts in vitro with a purified PLP-dependent apo-enzyme, to form the active holo-enzyme.

We have chosen Escherichia coli and its serine hydroxymethyltransferase (eSHMT) (foldtype I) as our model system because of: i) the known intracellular distribution and concentration of the B 6 vitamers in E. coli [START_REF] Fu | Distribution of B6 vitamers in Escherichia coli as determined by enzymatic assay[END_REF]; ii) the knowledge of the folding mechanism of eSHMT and its relationship to the addition of PLP [START_REF] Cai | The affinity of pyridoxal 5'-phosphate for folding intermediates of Escherichia coli serine hydroxymethyltransferase[END_REF][START_REF] Cai | Structural studies on folding intermediates of serine hydroxymethyltransferase using fluorescence resonance energy transfer[END_REF][START_REF] Cai | Structural studies on folding intermediates of serine hydroxymethyltransferase using single tryptophan mutants[END_REF][START_REF] Fu | Role of proline residues in the folding of serine hydroxymethyltransferase[END_REF]; iii) the availability of a number of important site-specific mutants of eSHMT; iv) the published high resolution structure of the holo-enzyme [START_REF] Scarsdale | Crystal structure at 2.4 A resolution of E. coli serine hydroxymethyltransferase in complex with glycine substrate and 5-formyl tetrahydrofolate[END_REF].

The mechanism of addition of PLP to several different fold-type I apo-enzymes has been previously investigated for aspartate aminotransferase, glutamate decarboxylase and tryptophanase [START_REF] Churchich | Reconstitution of aspartate aminotransferase from pig heart[END_REF][START_REF] Verge | Interactions between apoaspartate aminotransferase and pyridoxal 5'-phosphate. A stopped-flow study[END_REF][START_REF] Toney | Kinetics and equilibria for the reactions of coenzymes with wild type and the Y70F mutant of Escherichia coli aspartate aminotransferase[END_REF][START_REF] O'leary | Kinetics and mechanism of the binding of pyridoxal 5'-phosphate to apoglutamate decarboxylase. Evidence for a rate-determining conformation change[END_REF][START_REF] Erez | Pyridoxal phosphate binding to wild type, W330F, and C298S mutants of Escherichia coli apotryptophanase: unraveling the cold inactivation[END_REF]. These studies suggested a three-step mechanism, but were unable to identify unambiguously the structure of the intermediates. Since these studies were performed, advances in rapid scanning spectrophotometry and in the knowledge of folding mechanisms have provided a new impetus to further research efforts.

The folding mechanism of eSHMT has been investigated in detail and is understood better than that of any other fold-type I PLP-dependent enzyme. It may be divided into two phases. In the first, relatively rapid phase, two domains fold into their native state, forming an intermediate that has virtually all of the native secondary structure, exhibits most of the native tertiary structure, is a dimer, but is not able to bind PLP. In this intermediate, the N-terminus and an inter-domain segment remain exposed to solvent. In the slow phase, the intermediate is converted into the apo-enzyme, which is able to bind PLP. Inspection of the crystal structure of the holo-enzyme shows that PLP is buried in a deep cavity with no obvious route of entrance or escape to the solvent [START_REF] Scarsdale | Crystal structure at 2.4 A resolution of E. coli serine hydroxymethyltransferase in complex with glycine substrate and 5-formyl tetrahydrofolate[END_REF]. Thus, in the apo-enzyme the active site entrance must be more accessible than in the holo-enzyme.

The present work determines the mechanism of addition of PLP to apo-eSHMT and the absorption spectra of all kinetic species involved. The effect of the equilibrium between hydrate and aldehyde cofactor on the kinetics of holo-enzyme formation is also determined. Future studies will focus on cofactor specificity and the structure of the protein in all steps of the proposed mechanism. These will use eSHMT site-specific mutants and aldimine complexes of PLP with substrate and non-substrate amino acids.

EXPERIMENTAL PROCEDURES

Materials -Ingredients for bacterial growth were from Sigma-Aldrich. Chemicals for the purification of the enzymes were from BDH; DEAE-Sepharose and Phenyl-Sepharose from GE Healthcare. Alcohol dehydrogenase was from Sigma-Aldrich. eSHMT and methylenetetrahydrofolate dehydrogenase were purified as previously described [20,21]. (6S) H 4 PteGlu, was a gift from Eprova AG, Schaffhausen, Switzerland. PLP was from Sigma-Aldrich (98% pure). All other reagents were from Sigma-Aldrich. Apo-eSHMT was prepared using L-cysteine as previously described [START_REF] Cai | The affinity of pyridoxal 5'-phosphate for folding intermediates of Escherichia coli serine hydroxymethyltransferase[END_REF]. The apo-enzyme was stored in 10% glycerol at -20 °C for no more than 3 days before use. The concentration of the apo-eSHMT is calculated from an extinction coefficient at 278 nm of 42,790 cm -1 M -1 . A small, residual fraction (about 5%) of holo-enzyme was always present, as judged by activity assays.

Measurement of PLP molecules bound per monomer of enzyme -

Apo-eSHMT samples (10 μM in 1 ml) containing PLP at various concentrations (including control samples in which PLP was absent) were prepared in 50 mM HEPES-NaOH buffer at pH 7.2, equilibrated at 30 °C for 30 min and then reduced with NaBH 4 (0.2 M). Reduction of PLP-enzyme Schiff base irreversibly links the cofactor to the protein [START_REF] Fischer | The reaction of sodium borohydrate with muscle phosphorylase[END_REF]. NaBH 4 was prepared as a concentrated solution (2 M) in 50 mM NaOH. After 30 min from NaBH 4 addition, samples were concentrated in centrifuge filters (Millipore, Amicon Ultra, cutoff 30 KDa) and diluted with HEPES buffer repeatedly, so as to eliminate small molecular weight molecules, including reduced free cofactor. The final samples were diluted to 1 ml and an absorption spectrum recorded. The A 335 /A 280 ratio was calculated, in order to normalise the absorbance of the reduced internal aldimine on the basis of protein concentration, and then divided by the ratio found for a holo-enzyme sample. The result gives the fraction of PLP molecules bound per monomer of enzyme.

Mass spectrometry analysis -Two apo-enzyme samples (10 µM in 1 ml) were incubated with either 40 µM or 600 µM PLP. A third identical control sample did not contain PLP. After 30 min, NaBH 4 (0.2 M) was added to the samples. Small size molecules were removed using centrifuge filters as explained in the previous paragraph. Proteolytic digestion of each sample (1.5 μg) was performed adding approximately 0.1 μg of modified porcine trypsin (Millipore) in a solution of 25 mM ammonium bicarbonate and incubated at 37°C overnight. MALDI-TOF analyses of each tryptic peptide mixture were performed in a Voyager-DETM STR instrument (Applied Biosystem, Framingham, MA, USA).

Activity assays, spectral and stopped-flow studies -The hydroxymethyltransferase activity

was measured with L-serine and H 4 PteGlu as substrates as previously described [START_REF] Schirch | Serine hydroxymethyltransferase from Escherichia coli: purification and properties[END_REF]. The rate of L-allo-threonine cleavage was measured by coupling the reaction with the reduction of the product acetaldehyde by NADH and alcohol dehydrogenase [24].

Absorbance spectra and steady-state kinetic studies were performed on a Hewlett-Packard 8453 diode array spectrophotometer. Fluorescence emission measurements at equilibrium were carried out at 20 °C with a Fluoromax-3 spectrofluorimeter (HORIBA Jobin-Yvon, France), using a 1-cm pathlength quartz cuvette. Pyridoxine 5'-phosphate (PNP) fluorescence emission spectra were recorded from 350 to 500 nm (1-nm sampling interval; 5 nm emission slit) with the excitation wavelength set at 325 nm (1 nm excitation slit); PNP in the cuvette was 2 μM, while apo-eSHMT varied between 0.085 and 6 μM. The dissociation constant for the binding equilibrium was calculated from a Scatchard plot [START_REF] Cornish-Bowden | Fundamentals of enzyme kinetics[END_REF]. Curve-fitting procedures and statistical analysis were done with Prism (GraphPad Software Inc., San Diego, CA, USA).

Stopped-flow (absorbance and fluorescence) experiments were performed with an

Applied Photophysics SX18 apparatus (Leatherhead, UK) equipped with a 1 cm optical path observation chamber. A 360 nm filter was applied when measuring fluorescence emission obtained exciting PNP at 325 nm. Transient spectroscopy experiments were performed with the same apparatus reconfigured with the photodiode-array accessory. All experiments were carried out in HEPES-NaOH buffer, pH 7.2, at 30 °C.

Chemical quenched-flow experiments -

The rapid kinetics of internal aldimine formation were analysed by means of quenched-flow experiments carried out on a BioLogic QFM-400 apparatus, using NaBH 4 as quencher. Borohydride is known to reduce rapidly and efficiently imines and aldehydes [26]. When the internal aldimine of PLP-dependent enzymes is reduced, the cofactor is irreversibly attached to the protein, giving an absorption spectrum with a band at around 330 nm [27].

The four syringes of the quenched-flow apparatus at 30 °C contained respectively, apo-eSHMT (120 μM) in 50 mM HEPES-NaOH buffer at pH 7.2, PLP (20 μM) in the same buffer, water and NaBH 4 (2 M) in 50 mM NaOH. In each experiment, equal volumes of apo-enzyme and PLP (100 μl) were mixed, filling a 200 μl "delay line". Then, water pushed 180 μl of the reaction mixture from the first mixer into the second mixer where it encountered 20 μl of the borohydride solution. The quenched reaction (200 μl) was collected in a vial. The ageing time of reactions was determined by the flow speed through the delay line. Five reactions were carried out for each single ageing time. In the experiments with ageing times longer than one second, the flow was interrupted for the desired time interval once the reaction mixture filled the "delay line", and then started again in order to quench the reaction.

After being quenched, reactions were left in the vials for 30 min, in order to dissolve the gas bubbles formed from reaction of borohydride with water. Subsequently, the five samples relative to each ageing time were pooled (1 ml final volume), concentrated in a centrifuge filter and diluted with 50 mM HEPES-NaOH buffer (pH 7.2) repeatedly, so as to eliminate the small molecular weight molecules, including the reduced free cofactor. In the last dilution step, buffer was added to obtain a final volume of 1ml and absorption spectra recorded. The concentration of the reduced internal aldimine was calculated as described above.

Control stopped-flow experiments were carried out in order to compare the rate of NaBH 4 reduction of holo-eSHMT and free PLP in the aldehyde form. The extrapolated half times of the reactions at 0.2 M NaBH 4 (which was the concentration used in the quenchedflow experiments) were calculated to be 0.1 and 3.4 ms, respectively for holo-eSHMT and aldehyde PLP.

Data analysis -Binding of PLP to the second site was analysed according to Eqn 1, in which N and K d stand, respectively, for the number of PLP molecules bound per monomer of enzyme and the apparent dissociation constant of the equilibrium. Equation 1[

] [ ] d K PLP PLP N + + = 1
Fit to Eqn. 2 returned an initial velocity of the reaction in the absence of exogenous PLP (v 0 ) of 90 ± 3 μM min -1 while the velocity at infinite PLP concentration (v inf ) was 31 ± 5 μM min -1 (at pH 7.2 and 30 °C; holo-enzyme concentration was 0.1 μM).

Equation 2 ( ) [ ] [ ] ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ + × - = I i K PLP PLP v v v v
3 ( ) [ ] [ ] ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ + × - + = A i K PLP PLP v v v v 0 inf 0
Equations 1, 2 and 3 are intuitively derived from the following relationship, that may be generally applied to binding equilibria in which the ligand (in our case PLP) is in large excess with respect to the protein [START_REF] Cornish-Bowden | Fundamentals of enzyme kinetics[END_REF]:

[ ] [ ] d K PLP PLP Y + = ;
Y is the fraction of enzyme monomers that bind the cofactor at the second binding site, [PLP] is the concentration of free cofactor and K d is the dissociation constant of the related equilibrium.

Eqn. 4 is the sum of three exponential processes [START_REF] Cornish-Bowden | Fundamentals of enzyme kinetics[END_REF]. Equation 4)

1 ( ) 1 ( ) 1 ( 0 0 0 t k t k t k c b a e C e B e A Abs ⋅ - ⋅ - ⋅ - - ⋅ + ⋅ - ⋅ + ⋅ - ⋅ = Δ
In order to evaluate the accuracy of the fit to the sum of either three or two exponential processes, we performed the "F" test and the analysis of the explained variance [28]. A comparison of the fits obtained with either two or three exponentials gave an "F" value of 2.2, that corresponds to a probability of about 5% (one tail; 19 and 14 degrees of freedom, respectively for three and two exponentials; analysis carried out on time courses obtained at the four highest apo-enzyme concentrations). Fits of the same set of data to either one, two or three exponentials gave variances, respectively, of 3.93×10 -5 , 3.90×10 -5 and 1.77×10 -5 , demonstrating a clear-cut decrease of the explained variance with three exponentials. Finally, the distribution of the fit residuals when less than three exponentials were used to fit the data was non-random. These results clearly indicate that data fit better to the sum of three exponentials.

On and off rate constants (k on and k off ) for PNP binding kinetics were derived according to Cornish-Bowden [START_REF] Cornish-Bowden | Fundamentals of enzyme kinetics[END_REF].

The differential spectra obtained from the rapid scanning experiments were analysed by the singular value decomposition algorithm (SVD) [START_REF] Henry | Singular value decomposition: application to analysis of experimental data[END_REF] using the software MATLAB (MathWorks Inc., South Natick, MA). The analysis was aimed at determining the number of independent spectral components involved in the reaction. Absorption spectra were placed in a rectangular (m×n) matrix A, each column being a spectrum and each row a time course of absorbance at a single wavelength. Matrix A was decomposed by SVD into the product of three matrices: A=U×S×V T , where U and V are orthogonal matrices, and S is a diagonal matrix, with its nonzero elements arranged in decreasing order. The columns of matrix U contain the "basis spectra", and those of the V matrix contain the time dependence of each "basis spectrum". The diagonal S matrix contains the singular values that quantify the relative importance of each corresponding vector in U. Each column of matrix U×S represents a pseudospectrum, i.e. the absorption of one of the spectroscopic components required to approximate the original data and only the first few columns are significant, the last ones having negligibly small values. The signal-to-noise ratio is indeed very high in the earliest columns of U and V, while the random noise is mainly accumulated in the latest columns.

The global analysis of rapid scanning stopped-flow data and the band shape analysis of absorption spectra were carried out using the software Scientist (Micromath, Saint Louis, MO, USA). Time traces extracted from the hybrid stopped-flow diode array set of data and the kinetics of formation of the internal aldimine were globally fitted by numerical integration to systems of differential equations describing all possible models (made by reversible, unimolecular steps) that would give four independent spectral components when analysed by SVD. Models were made by: i) three consecutive reactions; ii) three parallel reactions; iii) three reactions branching from a single species; iv) two consecutive reactions paralleled by a single reaction. All reasonable combinations of the initial concentrations of components (as well as combinations of components that corresponded to the internal aldimine) were tested.

In total, 19 different versions of the four models were tested. Because of the limited capability of the software employed, only nine different time traces (corresponding to the most representative wavelengths) were fitted at once. The capability of the models to fit the data was evaluated on the basis of the distribution of fit residuals. All models but the one made by three consecutive reactions gave clearly non-random residuals. Several different fitting sessions were then carried out according to Scheme I, so as to cover the entire range of wavelengths acquired (95 different wavelengths).We noticed that the parameters produced by the least squares minimization routine were somewhat dependent on the initial estimates. To overcome this problem, the set of time courses was simulated over a wide range of the variable parameters in order to map the variance s 2 in the parameter hyperspace (as suggested by P.R. Bevington [START_REF] Bevington | Data reduction and error analysis for the physical sciences[END_REF]). The best parameter sets obtained by this procedure were used as the initial estimates in the minimization routines. Often, but not always, different initial estimates converged to the same minimum. In the end at least two clearly different minima were obtained, and the lower one was chosen as our best description of the experimental data.

RESULTS

Evidence of a second PLP binding site -PLP is a very reactive aldehyde. Before we could analyse the mechanism of PLP addition to eSHMT, we had to make sure that we were observing PLP binding only at the active site. The absorption spectrum of PLP changes radically as its aldehyde group forms an aldimine with the ε-amino group of the active site lysine residue (K229 in eSHMT), yielding the internal aldimine. A comparison between the spectra of free cofactor and holo-eSHMT shows a shift of λ max from 388 nm to 422 nm, with the maximum absorbance change taking place at 435 nm (data not shown). Following the change in absorbance at 435 nm with a stopped-flow spectrophotometer at different apoenzyme/PLP ratios resulted in complex kinetics (data not shown). Importantly, the shape and amplitude of the absorbance change with a large excess of apo-enzyme (100 µM) over PLP (10 µM) were profoundly different from those obtained with a reversed stoichiometric ratio.

In particular, with excess PLP there was a significant increase of the amplitude, suggesting that PLP was adding to at least one additional site.

As a test of this hypothesis, at increasing concentrations of PLP the binding was stoichiometric, until a first site was saturated (Fig. 1, inset). At higher concentrations a second molecule of PLP bound, although with a much lower affinity (Fig. 1). The best fit of data relative to the holo-enzyme to Eqn. 1, which assumes that one molecule of PLP per enzyme monomer is already bound, yielded an apparent dissociation constant (K d ) of 230 ± 60 µM for the binding equilibrium of the second molecule of cofactor at pH 7.2 and 30 °C.

Binding of PLP at the second site had an inhibitory effect on the hydroxymethyltransferase activity (data not shown), with an inhibition constant (K I ), calculated by best fitting the data to Eqn. 2, of 250 ± 100 µM. On the other hand, binding of the second PLP resulted in an increase of the H 4 PteGlu-independent L-allo-threonine cleavage activity (data not shown). Fitting data to Eqn.

Single wavelength analysis of holo-eSHMT formation -

The finding of a second PLP binding site requires that the studies on the mechanism of addition of PLP be carried out with an excess of apo-enzyme over cofactor, to minimize complications in determining a kinetic pathway and to maintain pseudo-first-order kinetics. A stopped-flow investigation was carried out, mixing equal volumes of 20 µM PLP with apo-enzyme at several different subunit concentrations (584, 350, 210, 126, 76, 44 and 27.2 µM) and recording absorbance changes at 435 nm (Fig. 2). Kinetic traces resulting from the four higher apo-enzyme concentrations were virtually superimposable and well described by the sum of three exponential processes (Eqn. 4). The sum of two exponential processes did not adequately fit the data, as indicated by the "F" test and the analysis of variance.

Global fitting of the four kinetic traces to Eqn. 4 indicated that the faster exponential process corresponded to a lag phase with a rate constant k a = 23.8 ± 0.9 s -1 and an amplitude A 0 = -0.0074 ±. 0.0001 AU (Fig. 2, inset). This was followed by two exponential increases in absorbance at 435 nm, whose rate constants and amplitudes were k b = 3.44 ± 0.03 s -1 , B 0 = 0.0605 ± 0.0001 AU, k c = 0.53 ± 0.02 s -1 and C 0 = 0.0110 ± 0.0002 AU. At apo-enzyme concentrations lower than 126 µM (before mixing), absorbance changes were systematically slower. The observation that k a does not change above 126 µM subunit concentration rules out that the lag corresponds to the bimolecular binding step. It rather suggests that a rapid equilibrium, corresponding to the binding of PLP to the enzyme, was established in the deadtime of the stopped-flow instrument. Therefore, the lag phase indicates that a reaction intermediate precedes the formation of the 422 nm absorbing internal aldimine.

Rapid scanning analysis of holo-eSHMT formation -

The course of the reaction of PLP with apo-eSHMT was followed by acquiring spectra with the stopped-flow spectrophotometer in the rapid scanning configuration. Spectra (800) were acquired at 10 ms intervals over a period of 8 s after a 9 ms delay from stop of flow. A control experiment was performed using the same conditions, but with the instrument set up to measure absorbance at a single wavelength (435 nm). Comparison of absorbance changes at 435 nm recorded in the rapid scanning and single wavelength configurations revealed that, when spectra are acquired in the scanning configuration, the absorbance change is smaller and takes place with a different kinetic pattern (data not shown). Traces by the two methods are superimposable up to 1 second, but thereafter diverge. A difference in the single wavelength and rapid scanning methods is the much higher light intensity used in the latter. A repeat of the rapid scanning experiment at a lower light intensity showed that photo bleaching was occurring after 1 s of reaction (400 spectra were acquired at 16 ms intervals over a period of 6.4 s, after a 14 ms delay from stop of flow). However, the low intensity of light in this experiment yielded increased noise in the data. In order to overcome this problem a hybrid data set was created, gathering the first 25 spectra acquired with intense light (at 10 ms intervals, from 9 ms to 0.26 s after stop of flow) and 385 spectra acquired using low intensity light (at 16 ms intervals, from 0.27 s to 6.4 s after stop of flow) (Fig. 3).

Differential spectra of the hybrid data set (with respect to the last spectrum acquired) were analysed by singular value decomposition (SVD) [START_REF] Henry | Singular value decomposition: application to analysis of experimental data[END_REF]. The result of the analysis suggested that three independent spectral components were required to describe the experimental data (the S values for the first seven components were 1.36, 0.79, 0.26, 0.18, 0.16, 0.15 and 0.14). Considering that a fourth spectral component has to be added because the analysis was carried out on differential spectra, this interpretation agrees with the single wavelength studies at 435 nm, described by the sum of three exponential processes.

Two additional experiments, that used intense light, were carried out in which either apo-enzyme (120 µM) or PLP (20 µM) were mixed with equal volumes of buffer. No spectral changes were observed in either experiment over a 6 s time period. These experiments gave the spectra of PLP and apo-enzyme after mixing (dashed line in Fig. 3). PLP in solution is an equilibrium of several different ionic and tautomeric structures with unique absorption properties. At pH 7.2 PLP is mainly present with its ring in the neutral form (about 5% of the cofactor is in the anionic form). Both dipolar ionic (where N 1 is protonated and O 3 ' is negative) and uncharged tautomers of the neutral ring exist, and each as both aldehyde and hydrate, although the uncharged hydrate is present as a negligible fraction [START_REF] Harris | Band-shape analysis and resolution of electronic spectra of pyridoxal phosphate and other 3-hydroxypyridine-4-aldehydes[END_REF]. The spectrum of PLP did not change significantly in the 9 ms after mixing with apoenzyme (Fig. 3). We conclude that the rapid-binding equilibrium established in the dead-time of the stopped-flow instrument does not alter the distribution of PLP ionic and tautomeric forms. Spectral resolution with log-normal curves permits a quantitative description of equilibria for hydration and tautomerization [START_REF] Metzler | Spectra of 3hydroxypyridines. Band-shape analysis and evaluation of tautomeric equilibria[END_REF][START_REF] Johnson | Analyzing spectra of vitamin B6 derivatives[END_REF]. The control spectrum of the cofactor after mixing with buffer (which corresponds to the spectrum of PLP at time zero of reaction) was analysed fitting the experimental data points to the sum of the expected component bands, using Metzler's equation. The parameters returned by the fitting were within ± 5% of the values found in the literature. Using the extinction coefficients found in the literature, the analysis showed that the uncharged aldehyde (λ max corresponding to 350 nm) was present as a 28% fraction, while the dipolar ionic aldehyde (388 nm) and the hydrate (323 nm) were present as 52% and 16% fractions, respectively. These values are in agreement with literature values for neutral PLP. A fourth band with an absorbance maximum at around 302 nm had to be included in the calculations in order to obtain a satisfactory fit and may account for the Biochemical Journal Immediate Publication. Published on 6 Mar 2007 as manuscript BJ20061681 © 2007 Biochemical Society remaining 4% of the cofactor. This component may be attributed to impurities and to a minor part to the cofactor being in the anionic form, which absorbs at this position. We do not know the exact nature of such impurities, however, the low absorbance maximum may indicate that the carbonyl group is either absent or saturated (and therefore not reactive).

The spectrum of the reaction acquired after 20 ms from stop of flow was also deconvoluted assuming that the absorption bands given by the hydrate form (323 nm) and the material absorbing at 302 nm were unchanged (all related parameters were therefore fixed in the fitting). Only the amplitudes of the bands corresponding to the aldehyde form of the cofactor (350 nm and 388 nm) were left free to vary during the fitting, while the other parameters were fixed to the values found in the previous deconvolution. A fifth absorption band with a maximum at 335 nm had to be included in order to obtain a good fit. This corresponds to a reaction intermediate generated in the very early phase of the reaction. The amplitudes of both bands relative to the aldehyde form of PLP (350 nm and 388 nm) decreased. This analysis suggested that only the aldehyde form of PLP reacts in the first few milliseconds of the reaction, giving the intermediate that absorbs at 335 nm. The kinetics obtained fitted well to the sum of two exponential processes with rate constants of 38 ± 16 s -1 and 0.79 ± 0.15 s -1 and amplitudes of 3.1 ± 0.5 μM and 6.4 ± 0.4 μM, respectively (Fig. 4). A constant 0.5 µM internal aldiminine had to be added to account for a small amount of holo-enzyme present as a contaminant in the apo-enzyme samples. Taking into account the large standard deviation of the two rate constants, their values roughly correspond to k a (23.8 ± 0.9 s -1 ) and k c (0.53± 0.02 s -1 ) of absorbance changes measured at 435 nm in the stopped-flow experiments.

Quenched-flow analysis

Global analysis -Any kinetic scheme describing the reaction between apo-enzyme and PLP should be consistent with the results of the SVD analysis of the rapid scanning data, which indicated the presence of four independent spectral components. To begin with, we tested all kinetic models, made by reversible unimolecular steps, that satisfy this requirement, regardless to their consistency with the chemistry of the system under study. Single wavelength traces from the hybrid diode array data and the kinetics of internal aldimine formation were globally fitted to all models. Because of the limits of the software, no more than nine time traces (chosen among the most representative ones) could be fitted at once.

Only the model corresponding to three consecutive reactions fitted the data.

A three-step model that is consistent with both our data and the expected mechanism of the reaction is represented by Scheme I, in which PLP Ald and PLP Hyd are the cofactor in the aldehyde and hydrate forms; apoE•PLP Ald and apoE•PLP Hyd are the related non-covalent complexes with the apo-enzyme; E=PLP* is an intermediate internal aldimine and holoE is the holo-enzyme. Fitting data to Scheme I (several fitting sessions were carried out so as to cover the entire range of wavelengths acquired), we assumed that, when the stopped-flow registration of the reaction begins only the species in square brackets are observable and the concentrations of the non-covalent complexes (apoE•PLP Hyd and apoE•PLP Hyd ) correspond to those of their related free PLP forms. This assumption is justified by the saturation behaviour observed in the single wavelength experiments (Fig. 2) and the results of band shape analyses.

Concentrations of apoE•PLP Hyd and apoE•PLP Ald at time zero were respectively, 1.6 and 8 μM, while those of E=PLP* and holoE were nil. In the global fittings, the concentration of internal aldimine was made equal to the sum of E=PLP* and holoE. The absorbance at any wavelength corresponded to the sum of the concentrations of each of the four kinetic species multiplied by their micromolar extinction coefficient at that wavelength, plus a constant absorbance contributed by the apo-enzyme and the band at 302 nm (found in the band shape analyses and assumed to be mostly inert material; the analysis would have detected any incongruence with this assumption in terms of a poor fit at wavelengths around 302 nm). In the fitting, the extinction coefficients of apoE•PLP Hyd and apoE•PLP Ald were fixed at the values calculated in the band shape analysis of the free cofactor, while those of E=PLP* and holoE were free to vary between zero and infinity. Under the experimental conditions used, either the second or the third reaction (or both) of Scheme I must be virtually irreversible. This hypothesis is confirmed by the observation that all cofactor binds covalently to the apoenzyme (Fig. 4). The global fit was therefore carried out with three different versions of Scheme I. Only the one in which the third reaction was irreversible fitted the data (Fig. 5), returning the following rate constants: k 1 = 1.2 s -1 , k -1 = 0.3 s -1 , k 2 = 18 s -1 , k -2 = 6.5 s -1 and k 3 = 3.5 s -1 .

The absorption spectra of the kinetic species, reconstructed from the micromolar extinction coefficients estimated in the fitting, are shown in Fig. 6. The spectrum of the intermediate E=PLP* was deconvoluted into its component bands using Metzler's band shape analysis method (Fig. 7). The main component band has its maximum absorbance at 386 nm.

Two additional bands with maxima at 337 and 286 nm, respectively, had to be included in order to obtain a good fit. The other parameters returned by the fitting were within a ± 5% range of the values found in the literature for absorption spectra of PLP-dependent enzymes [START_REF] Metzler | Quantitative description of absorption spectra of a pyridoxal phosphate-dependent enzyme using lognormal distribution curves[END_REF].

Binding of pyridoxine 5'-phosphate to apo-eSHMT -Scheme I assumes that hydrate PLP

binds rapidly to apo-eSHMT. This hypothesis was tested by following the binding of the structural analogue pyridoxine 5'-phosphate (PNP), which can not proceed further in the reaction sequence because it lacks an aldehyde. PNP does bind to apo-eSHMT, as indicated by the increase of this vitamer's fluorescence emission observed upon mixing with the apoenzyme (data not shown). We calculated a K d = 90 ± 5 nM for this binding equilibrium.

Stopped-flow fluorescence experiments in which 2 μM PNP was mixed with excess apoenzyme (60 μM), so as to guarantee pseudo-first order conditions, gave biphasic kinetics with apparent rate constants of 178 ± 4 s -1 and 3.21 ± 0.01 s -1 (data not shown). The linear dependence of the faster rate constant on protein concentration indicated that it corresponds to the encounter between PNP and apo-eSHMT. The much slower phase that follows is likely to be a conformational change of the enzyme. A k on = 1.6 ± 0.4 s -1 μM -1 and a k off = 76 ± 14 s -1 were calculated for the bimolecular step. With 60 μM apo-enzyme (as in the rapid scanning experiments) an observed binding rate constant of about 172 s -1 would be measured (k on × 60 μM + 76 s -1 ). This means that binding takes place with a half time of about 4 ms, which is not far from the dead time of the stopped-flow instrument (2-3 ms).

DISCUSSION

Second PLP binding site -The apparent dissociation constants calculated in the binding, inhibition and activation experiments are very similar. This suggests that they are relative to the same binding equilibrium. K346, the residue that binds the second PLP molecule, is highly conserved in SHMTs and forms a salt-bridge with the carboxylate group of the glutamyl residue of H 4 PteGlu [START_REF] Fu | Location of the pteroylpolyglutamate-binding site on rabbit cytosolic serine hydroxymethyltransferase[END_REF]. PLP binding at this site therefore inhibits the folatedependent serine cleavage reaction. The activation of the folate-independent L-allo-threonine cleavage is analogously explained, assuming that PLP binding to K346 mimics H 4 PteGlu binding. It was indeed reported by Webb and Matthews [START_REF] Webb | 4-Chlorothreonine is substrate, mechanistic probe, and mechanism-based inactivator of serine hydroxymethyltransferase[END_REF] that H 4 PteGlu stimulates the aldol cleavage reaction. To our knowledge, this second PLP binding site has not been reported previously. Our discovery was incidental and we limited its significance to the design of the kinetic experiments.

Proposed mechanism for PLP addition to apo-eSHMT -Before the reaction begins, the free cofactor is present as an equilibrium mixture of hydrate and aldehyde forms (PLP Hyd and PLP Ald in Scheme I). Upon mixing PLP and apo-enzyme, a rapid non-covalent binding equilibrium is established in the dead-time of the stopped-flow instrument. Scheme I assumes that both hydrate and aldehyde PLP binds rapidly to the apo-enzyme. This assumption is well substantiated. The solution rate constant for interconversion of PLP Hyd and PLP Ald at pH 7.0 and 25 °C has been reported to be around 10 s -1 [START_REF] Ahrens | Kinetic study of the hydration mechanism of vitamin B6 and related compounds[END_REF]. We found it to be 16 s -1 in the buffer used for our experiments at pH 7.2 and 30 °C (data not shown). This rate constant is not observed in either the single wavelength or rapid scanning kinetic results in our binding studies. This suggests that hydrate PLP binds to the enzyme. Moreover, we showed that PNP, a good structural analogue of hydrate PLP, binds rapidly to the apo-enzyme.

The rate constants determined for Scheme I from the rapid scanning data compare favourably with those obtained for the single wavelength studies. The values of k 1 , k -1 k 2 , k -2 and k 3 make it clear that the complex of the enzyme with PLP Ald (apoE•PLP Ald ) reacts much faster than the corresponding complex with the hydrate (apoE•PLP Hyd ). Thus, upon combination of the cofactor with the apo-enzyme, the apoE•PLP Ald complex evolves to the equilibrium species (E=PLP*) before apoE•PLP Hyd decays to any significant extent.

Therefore, the apparent rate constants k a (23.8 s -1 ) and k b (3.44 s -1 ) from the single wavelength studies roughly correspond, respectively, to the sum of the intrinsic rate constants k 2 +k -2 (18 s - 1 + 6.5 s -1 ) and to k 3 (3.5 s -1 ). The slowest exponential process, dominated by k c (0.53 s -1 ), represents the decay of apoE•PLP Hyd , which corresponds to the conversion of the enzymebound hydrate PLP (apoE•PLP Hyd ) to the enzyme-bound aldehyde form (apoE•PLP Ald ).

Alternatively, it may correspond to the dissociation of hydrate PLP from the enzyme, its dehydration and binding to the apo-enzyme as aldehyde.

The absorbance change at 435 nm reports the formation of the holo-enzyme (holoE).

There is a pronounced lag in the formation of holoE in the stopped-flow studies (Fig. 2, inset).

Within experimental error, the rate of the burst in the quench-flow studies (Fig. 4) is the same as the lag in the rapid reaction studies. This suggests that holoE is formed from an intermediate aldimine species not absorbing at 435 nm (shown as E=PLP* in Scheme I).

The spectrum of E=PLP* has a major absorption band with a maximum at 386 nm (Fig. 7). Protonated aldimines are normally observed to have maximum absorption between 410 nm and 430 nm. However, aldimines absorbing between 385 and 400 nm have been reported in the literature [START_REF] Minelli | Reactions of DOPA (3,4dihydroxyphenylalanine) decarboxylase with DOPA[END_REF][START_REF] Hayashi | Rat liver aromatic L-amino acid decarboxylase: spectroscopic and kinetic analysis of the coenzyme and reaction intermediates[END_REF][40]. In particular, evidence was presented that in the aspartate aminotransferase Y225F mutant at high pH, the chromophore absorbing maximally at 386 nm was the unprotonated internal aldimine [START_REF] Goldberg | Structure of the complex between pyridoxal 5'-phosphate and the tyrosine 225 to phenylalanine mutant of Escherichia coli aspartate aminotransferase determined by isotope-edited classical Raman difference spectroscopy[END_REF][START_REF] Goldberg | The tyrosine-225 to phenylalanine mutation of Escherichia coli aspartate aminotransferase results in an alkaline transition in the spectrophotometric and kinetic pKa values and reduced values of both kcat and Km[END_REF]. In this case, the maximum absorbance of the unprotonated aldimine, usually positioned at around 360 nm, was shifted to higher wavelengths due to the alteration of the active site environment caused by the mutation. The unusual absorption maximum at 386 nm of the E=PLP* aldimine suggests that the incompletely formed active site of apo-eSHMT may also bind PLP as an unprotonated aldimine. A smaller absorption peak is also observed at 337 nm for E=PLP* (Fig. 7). This may be attributed to an enolimine tautomer, or more likely to a required carbinolamine intermediate that is in rapid equilibrium with the aldimine [START_REF] Kallen | Chemical and spectroscopic properties of pyridoxal and pyridoxamine phosphates[END_REF].

The final step in Scheme I (E=PLP* going to holo-E) is most likely a conformational change. This step is predicted to be largely irreversible, trapping the PLP in its deep cleft as revealed from its crystal structure. Interestingly, the slower fluorescence change associated to PNP binding to apo-eSHMT takes place with a similar rate constant, suggesting that the final conformational change may be independent from the internal aldimine formation.

Our future studies will be aimed at answering several key questions raised by the proposed mechanism shown in Scheme I. The first one concerns the structural features the cofactor requires in order to form the first non-covalent complex with the apo-enzyme and then trigger the conformational change that yields the holo-enzyme. Do aldimines of PLP with amino acids bind? Does eSHMT bind the aldimines of its substrates glycine and serine selectively with respect to aldimines of non-substrate amino acids? What is the K d of the first binding equilibrium? Logic would predict that the first complex formed by the apo-enzyme with PLP would result in a relatively low K d , so that PLP would not be sequestered by the cell cytoplasm, where this very reactive aldehyde is subject to reactions with a host of nucleophiles. In the cell there may be little PLP Ald . Most of it is likely present as aldimines with amino acids or complexes with thiol compounds [START_REF] Fu | Distribution of B6 vitamers in Escherichia coli as determined by enzymatic assay[END_REF]. Studies on PNP binding gave a K d of about 47 µM for the first binding equilibrium of (k off /k on ).

Other interesting questions relate to the role of K229 in the final conformational change (E=PLP* going to holo-E) and the structure of apo-eSHMT. The first one may be answered by studies on site-specific mutants. Folding studies suggest that an N-terminal segment and an interdomain loop that form part of the active site are both exposed to solvent in a folding intermediate that precedes the formation of apo-eSHMT. May be these structural elements of the protein are still exposed in apo-eSHMT and E=PLP*.

  to Eqn. 3 gave an initial velocity in the absence of exogenous PLP of 45 ± 5 μM min - 1 and a velocity at infinite PLP concentration of 72 ± 6 μM min -1 (at pH 7.2 and 30 °C; holoenzyme concentration was 1.25 μM).

Equation

  3 returned an activation constant (K A ) of 230 ± 40 µM. The enzyme residue involved in the covalent binding of PLP to the second site was identified by mass spectrometry analysis of samples of apo-enzyme (10 µM) incubated with either 40 µM or 600 µM PLP. The compared analysis of tryptic peptides showed that the cofactor was bound to Lys 229 in the sample with 40 µM PLP and to both Lys 229 and Lys 346 in the sample with 600 µM PLP.

-

  The formation of the internal aldimine between the PLP aldehyde group and the Lys229 ε-amino group was monitored by chemical quenched-flow experiments in which equal volumes of apo-enzyme (120 µM) and cofactor (20 µM) were rapidly mixed and the reaction quenched at different time intervals with concentrated NaBH 4 (0.2 M). This experiment correctly reports the formation of the internal aldimine only if the aldehyde PLP and the internal aldimine are reduced by NaBH 4 much more rapidly than the internal aldimine is formed. This point was checked by stopped-flow control experiments, which used either holo-eSHMT or free PLP.

FIGURE 1 .

 1 FIGURE 1. Equilibrium binding of PLP to eSHMT. The number of PLP molecules bound per monomer of enzyme (10 µM) was measured as a function of cofactor concentration in 50 mM HEPES-NaOH buffer, pH 7.2, at 30 ºC. The continuous line through the experimental points (○, apo-enzyme; •, holo-enzyme) derived from the best fit of data to Eqn 1. The inset shows the different behaviour of apo-and holo-enzyme at low PLP concentration.

FIGURE 2 .

 2 FIGURE 2. Absorbance changes at 435 nm observed upon stopped-flow mixing apo-enzyme and PLP. Absorbance changes at 435 nm upon stopped-flow mixing equal volumes of 584 µM, 350 µM, 210 µM, 126 µM (⎯⎯), 76 µM (-----), 44 µM (⋅⋅⋅⋅⋅⋅⋅), 27.2 µM (-⋅-⋅-⋅-⋅) apo-enzyme and 20 µM PLP. Conditions were as in Fig.1. The inset shows the lag phase.

FIGURE 3 .

 3 FIGURE 3. Spectral changes observed upon stopped-flow mixing 120 μM apoenzyme and 20 μM PLP. Only spectra taken at 20 ms intervals (after 9 ms from stop of flow) with intense light and at 64 ms intervals with low intensity light are shown for clarity. The sum of the spectra of PLP and apo-enzyme obtained after mixing with buffer is shown as a thick, dashed line. Conditions were as in Fig.1.

FIGURE 4 .

 4 FIGURE 4. Kinetics of Schiff base formation between 10 µM PLP and 60 µM apoenzyme, using chemical quenched-flow. The experimental points represent covalently bound PLP and the theoretical line is from fitting to the sum of two exponential processes.Conditions were as in Fig.1. The inset shows the burst in the early phase of the reaction.

FIGURE 5 .

 5 FIGURE 5. Representative results of the global fit of absorbance changes and internal aldimine formation. Upper panel -Internal aldimine formation (symbols) and nine kinetic traces (only three of which are shown for clarity in the inset) were fitted by least squares to differential equations corresponding to Scheme I. Middle panel -Expanded view showing the early phase of the reaction. Lower panel -residuals of the fit relative to all nine kinetic traces and the internal aldimine formation.

FIGURE 6 .

 6 FIGURE 6. Absorption spectra of kinetic species in Scheme I, as reconstructed from the estimated extinction coefficients yielded by the global fit. apo-E•PLP Hyd (⎯⎯), apo-E•PLP Ald (-----), E=PLP* (⋅⋅⋅⋅⋅) and holo-E (⋅-⋅-⋅-).

FIGURE 7 .

 7 FIGURE 7. Band shape analysis of the absorption spectrum of the intermediate holoenzyme. The reconstructed spectrum of E=PLP* (×) was deconvoluted into its component bands (continuous lines) with absorption maxima at 386 nm, 337 nm and 286 nm.The inset shows the residuals of the fit.

  Figure 5

Biochemical Journal Immediate Publication. Published on 6 Mar 2007 as manuscript BJ20061681 © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 6 Mar 2007 as manuscript BJ20061681 © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 6 Mar 2007 as manuscript BJ20061681 © 2007 Biochemical Society

© 2007 Biochemical Society

ACKNOWLEDGEMENTS

We thank Professor Verne Schirch for his precious help in planning, discussing the experiments and help in writing the manuscript. We also thank Amleto Ballini for his technical support and Renata Quattrini, an undergraduate student participant. We are grateful to Eprova AG, Schaffhausen, Switzerland, for providing pure (6S) H 4 PteGlu. This work was supported by grants of the Italian Ministero dell'Università e della Ricerca.