Isotope effect studies of the pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii

The pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii shows a nitrogen isotope effect k14/k15 = 0.9770 +/- 0.0021, a carbon isotope effect k12/k13 = 1.0308 +/- 0.0006, and a carbon isotope effect for L-[alpha-2H]histidine of 1.0333 +/- 0.0001 at pH 6.3, 37 degrees C. These results indicate that the overall decarboxylation rate is limited jointly by the rate of Schiff base interchange and by the rate of decarboxylation. Although the observed isotope effects are quite different from those for the analogous glutamate decarboxylase from Escherichia coli [Abell, L. M., & O'Leary, M. H. (1988) Biochemistry 27, 3325], the intrinsic isotope effects for the two enzymes are essentially the same. The difference in observed isotope effects occurs because of a roughly twofold difference in the partitioning of the pyridoxal 5'-phosphate-substrate Schiff base between decarboxylation and Schiff base interchange. The observed nitrogen isotope effect requires that the imine nitrogen in this Schiff base is protonated. Comparison of carbon isotope effects for deuteriated and undeuteriated substrates reveals that the deuterium isotope effect on the decarboxylation step is about 1.20; thus, in the transition state for the decarboxylation step, the carbon-carbon bond is about two-thirds broken.     

Nitrogen isotope effects on glutamate decarboxylase from Escherichia coli

The nitrogen isotope effect on the decarboxylation of glutamic acid by glutamate decarboxylase from Escherichia coli has been measured by comparison of the isotopic composition of the amino nitrogen of the product gamma-aminobutyric acid isolated after 10-20% reaction with that of the starting glutamic acid. At pH 4.7, 37 degrees C, the isotope effect is k14/k15 = 0.9855 +/- 0.0006 when compared to unprotonated glutamic acid. Interpretation of this result requires knowledge of the equilibrium nitrogen isotope effect for Schiff base formation. This equilibrium isotope effect is k14/k15 = 0.9824 for the formation of the unprotonated Schiff base between unprotonated valine and salicylaldehyde. Analysis of the nitrogen isotope effect on decarboxylation of glutamic acid and of the previously measured carbon isotope effect on this same reaction [O'Leary, M.H., Yamada, H., & Yapp, C.J. (1981) Biochemistry 20, 1476] shows that decarboxylation and Schiff base formation are jointly rate limiting. The enzyme-bound Schiff base between glutamate and pyridoxal 5'-phosphate partitions approximately 2:1 between decarboxylation and return to the starting state. The nitrogen isotope effect also reveals that the Schiff base nitrogen is protonated in this intermediate.     

Aspartate beta-decarboxylase from Alcaligenes faecalis: carbon-13 kinetic isotope effect and deuterium exchange experiments

We have measured the 13C kinetic isotope effect at pH 4.0, 5.0, 6.0, and 6.5 and in D2O at pD 5.0 and the rate of D-H exchange of the alpha and beta protons of aspartic acid in D2O at pD 5.0 for the reaction catalyzed by the enzyme aspartate beta-decarboxylase from Alcaligenes faecalis. The 13C kinetic isotope effect, with a value of 1.0099 +/- 0.0002 at pH 5.0, is less than the intrinsic isotope effect for the decarboxylation step, indicating that the decarboxylation step is not entirely rate limiting. We have been able to estimate probable values of the relative free energies of the transition states of the enzymatic reaction up to and including the decarboxylation step from the 13C kinetic isotope effect and the rate of D-H exchange of alpha-H. The pH dependence of the kinetic isotope effect reflects the pKa of the pyridine nitrogen of the coenzyme pyridoxal 5'-phosphate but not that of the imine nitrogen. A mechanism is proposed for the exchange of aspartate beta-H that is consistent with the stereochemistry suggested earlier.     

Lysine-313 of 5-aminolevulinate synthase acts as a general base during formation of the quinonoid reaction intermediates

5-Aminolevulinate synthase catalyzes the condensation of glycine and succinyl-CoA to form CoA, carbon dioxide, and 5-aminolevulinate. This represents the first committed step of heme biosynthesis in animals and some bacteria. Lysine 313 (K313) of mature murine erythroid 5-aminolevulinate synthase forms a Schiff base linkage to the pyridoxal 5'-phosphate cofactor. In the presence of glycine and succinyl-CoA, a quinonoid intermediate absorption is transiently observed in the visible spectrum of purified murine erythroid ALAS. Mutant enzymes with K313 replaced by glycine, histidine, or arginine exhibit no spectral evidence of quinonoid intermediate formation in the presence of glycine and succinyl-CoA. The wild-type 5-aminolevulinate synthase additionally forms a stable quinonoid intermediate in the presence of the product, 5-aminolevulinate. Only conservative mutation of K313 to histidine or arginine produces a variant that forms a quinonoid intermediate with 5-aminolevulinate. The quinonoid intermediate absorption of these mutants is markedly less than that of the wild-type enzyme, however. Whereas the wild-type enzyme catalyzes loss of tritium from [2-3H2]-glycine, mutation of K313 to glycine results in loss of this activity. Titration of the quinonoid intermediate formed upon binding of 5-aminolevulinate to the wild-type enzyme indicated that the quinonoid intermediate forms by transfer of a single proton with a pK of 8.1 +/- 0.1. Conservative mutation of K313 to histidine raises this value to 8.6 +/- 0.1. We propose that K313 acts as a general base catalyst to effect quinonoid intermediate formation during the 5-aminolevulinate synthase catalytic cycle.     

13C NMR spectroscopy of labeled pyridoxal 5'-phosphate. Model studies, D-serine dehydratase, and L-glutamate decarboxylase.

Pyridoxal 5'-phosphate labeled to the extent of 90% with 13C in the 4' (aldehyde) and 5' (methylene) positions has been synthesized. 13C NMR spectra of this material and of natural abundance pyridoxal 5'-phosphate are reported, as well as 13C NMR spectra of the Schiff base formed by reaction of pyridoxal 5'-phosphate with n-butylamine, the secondary amine formed by reduction of this Schiff base, the thiazolidine formed by reaction of pyridoxal 5'-phosphate with cysteine, the hexahydropyrimidine formed by reaction of pyridoxal 5'-phosphate with 1,3-diaminobutane, and pyridoxamine 5'-phosphate. The range of chemical shifts for carbon 4' in these compounds is more than 100 ppm, and thus this chemical shift is expected to be a sensitive indicator of structure in enzyme-bound pyridoxal 5'-phosphate. The chemical shift of carbon 5', on the other hand, is insensitive to these structure changes. 13C NMR spectra have been obtained at pH 7.8 and 9.4 for D-serine dehydratase (Mr = 46,000) containing natural abundance pyridoxal 5'-phosphate and containing 13C-enriched pyridoxal 5'-phosphate. The enriched material contains two new resonances not present in the natural abundance material, one at 167.7 ppm with a linewidth of approximately 24 Hz, attributed to carbon 4' of the Schiff base in the bound coenzyme, and one at 62.7 Hz with a linewidth of approximately 48 Hz attributed to carbon 5' of the bound Schiff base. A large number of resonances due to individual amino acids are assigned. The NMR spectrum changes only slightly when the pH is raised to 9.4. The widths of the two enriched coenzyme resonances indicate that the coenzyme is rather rigidly bound to the enzyme but probably has limited motional freedom relative to the protein. 13C NMR spectra have been obtained for L-glutamate decarboxylase containing natural abundance pyridoxal 5'-phosphate and 13C-enriched pyridoxal 5'-phosphate. Under conditions where the two enriched 13C resonances are clearly visible in D-serine dehydratase, no resonances are visible in enriched L-glutamate decarboxylase, presumably because the coenzyme is rigidly bound to the protein and the 300,000 molecular weight of this enzyme produces very short relaxation times for the bound coenzyme and thus very broad lines.     

The pyridoxal 5' -phosphate site in rabbit skeletal muscle glycogen phosphorylase b: an ultraviolet and 1H and 31P nuclear magnetic resonance spectroscopic study.

1 H NMR spectra of the 3-0-methylpyridoxal 5'-phosphate-n-butylamine reaction product indicated that this analogue forms a Schiff base in aprotic solvent. The uv spectral properties of 3-0-methylpyridoxal-5'-phosphate phosphorylase b correspond to those of the n-butylamine Schiff base derivative in dimethyl sulfoxide. On the basis of that and auxiliary uv and 1H NMR spectra of pyridoxal and pyridoxal 5'-phosphate and the corresponding Schiff base derivatives we have verified that pyridoxal 5' -phosphate is also bound as a Schiff base to phosphorylase and not as an aldamine. Since 3-0-methylpyridoxal-5'-phosphate phosphorylase is active, a proton shuttle between the 3-hydroxyl group and the pyridine nitrogen is excluded. This directs attention to the 5' -phosphate group of the cofactor as a candidate for a catalytic function. 31P NMR spectra of pyridoxal 5' -phosphate in phosphorylase b indicated that deprotonation of the 5' -phosphate group was unresponsive to external pH. Interaction of phosphorylase b with adenosine 5' -monophosphate, the allosteric effector required activity, and arsenate, which substitutes for phosphate as substrate, triggered a conformational change which resulted in deprotonation of the 5' -phosphate group of pyridoxal 5' at pH 7.6. It now behaved like in the pyridoxal-phosphate-epsilon-aminocaproate Schiff base in aqueous buffer, where the diionized form is dominant at this pH. Differences of line widths of the adenosine 5' -monophosphate signal point to different life times of the allosteric effector- enzyme complexes in the presence and absence of substrate (arsenate).     

Study of the hydrolysis and ionization constants of Schiff base from pyridoxal 5''-phosphate and n-hexylamine in partially aqueous solvents. An application to phosphorylase b.

Formation and hydrolysis rate constants as well as equilibrium constants of the Schiff base derived from pyridoxal 5'-phosphate and n-hexylamine were determined between pH 3.5 and 7.5 in ethanol/water mixtures (3:17, v/v, and 49:1, v/v). The results indicate that solvent polarity scarcely alters the values of these constants but that they are dependent on the pH. Spectrophotometric titration of this Schiff base was also carried out. We found that a pKa value of 6.1, attributed in high-polarity media to protonation of the pyridine nitrogen atom, is independent of solvent polarity, whereas the pKa of the monoprotonated form of the imine falls from 12.5 in ethanol/water (3:17) to 11.3 in ethanol/water (49:1). Fitting of the experimental results for the hydrolysis to a theoretical model indicates the existence of a group with a pKa value of 6.1 that is crucial in the variation of kinetic constant of hydrolysis with pH. Studies of the reactivity of the coenzyme (pyridoxal 5'-phosphate) of glycogen phosphorylase b with hydroxylamine show that this reaction only occurs when the pH value of solution is below 6.5 and the hydrolysis of imine bond has started. We propose that the decrease in activity of phosphorylase b when the pH value is less than 6.2 must be caused by the cleavage of enzyme-coenzyme binding and that this may be related with protonation of the pyridine nitrogen atom of pyridoxal 5'-phosphate.     

Isotope effect studies of the role of metal ions in isocitrate dehydrogenase.

Pig heart NADP+-dependent isocitrate dehydrogenase requires a metal ion for activity. Under optimum conditions (pH 7.5, Mg2+ present), the carbon isotope effect is k12/k13 = 0.9989 +/- 0.0004 for the carboxyl carbon undergoing decarboxylation and hydrogen isotope effects are VmaxH/VmaxD = 1.09 +/- 0.04 and (Vmax/Km)H/(Vmax/Km)D = 0.76 +/- 0.12 with threo-D,L-[2-2H]isocitric acid. Deuterium isotope effects measured by the equilibrium perturbation technique under the same conditions are VH/VD = 1.20 for the forward reaction and 1.02 for the reverse reaction. Under these conditions the rate-determining step in the enzymatic reaction must be product release. Dissociation of isocitrate from the enzyme-isocitrate complex and the enzyme-NADP+ complex must be two or more orders of magnitude slower than the chemical steps. The catalytic activity of the enzyme is about tenfold lower in the presence of Ni2+ than in the presence of Mg2+. The carbon isotope effect in the presence of Ni2+ at pH 7.5 is k12/k13 = 1.0051 +/- 0.0012 and the hydrogen isotope effects are VmaxH/VmaxD = 0.98 +/- 0.07 and (Vmax/Km)H/(Vmax/Km)D = 1.11 +/- 0.14. Thus, the rate decrease caused by substitution of Ni2+ for Mg2+ must result from the effects of metal on substrate and product binding and dissociation, rather than effects of metal on catalysis. However, a more detailed analysis of the carbon isotope effects reveals that there is also a large metal effect on the rate of the decarboxylation step, consistent with the view that the carbonyl oxygen of the oxalosuccinate intermediate is coordinated to the metal during decarboxylation.     

Altering the reaction specificity of eukaryotic ornithine decarboxylase

Ornithine decarboxylase (ODC) catalyzes the first committed step in the biosynthesis of polyamines, and it has been identified as a drug target for the treatment of African sleeping sickness, caused by Trypanosoma brucei. ODC is a pyridoxal 5'-phosphate (PLP) dependent enzyme and an obligate homodimer. X-ray structural analysis of the complex of the T. brucei wild-type enzyme with the product putrescine reveals two structural changes that occur upon ligand binding: Lys-69 is displaced by putrescine and forms new interactions with Glu-94 and Asp-88, and the side chain of Cys-360 rotates into the active site to within 3.4 A of the imine bond. Mutation of Cys-360 to Ala or Ser reduces the k(cat) of the decarboxylation reaction by 50- and 1000-fold, respectively. However, HPLC analysis of the products demonstrates that the mutant enzymes almost exclusively catalyze a decarboxylation-dependent transamination reaction to form pyridoxamine 5-phosphate (PMP) and gamma-aminobutyraldehyde, instead of PLP and putrescine. This side reaction arises when the decarboxylated substrate intermediate is protonated at C4' of PLP instead of at the C(alpha) of substrate. For the reaction catalyzed by the wild-type enzyme, this side reaction occurs infrequently (<0.01% of the turnovers). Single turnover analysis and multiwavelength stopped-flow spectroscopic studies suggest that for the mutant ODCs protonation at C4' occurs either very rapidly or in a concerted reaction with decarboxylation and that the rate-limiting step in the steady-state reaction is Schiff base hydrolysis/product release. These studies demonstrate a role for Cys-360 in the control of the C(alpha) protonation step that catalyzes the formation of the physiological product putrescine. The results further provide insight into the mechanism by which this class of PLP-dependent enzymes controls reaction specificity.     

Kinetic and thermodynamic parameters for Schiff base formation of vitamin B6 derivatives with amino acids

Thermodynamic and kinetic parameters for Schiff base formation of pyridoxal 5'-phosphate and pyridoxal with epsilon-aminocaproic acid as well as of pyridoxal 5'-phosphate with L-serine were obtained in 0.1 M sodium pyrophosphate buffer as a function of temperature. Changes in enthalpy, which were determined by direct microcalorimetry, were small at 25 degrees C, but varied strongly with pH for the reaction of pyridoxal 5'-phosphate with the amino acids. In contrast to the fast Schiff base formation of pyridoxal 5'-phosphate, a very slow reaction was found for pyridoxal and epsilon-aminocaproic acid concomitant with a larger change in enthalpy. By preventing hemiacetal formation the phosphate moiety plays a crucial role.     

Inhibition of horse muscle acylphosphatase by pyridoxal 5'-phosphate.

It has been shown that horse muscle acylphosphatase is inhibited by pyridoxal 5'-phosphate and that the inhibition is pH dependent, reversible and competitive with respect to substrate binding. Spectral analysis on the EI complex demonstrates the presence of a Schiff base. Reduction of the pyridoxal 5'-phosphate-inhibited enzyme with sodium borohydride, followed by amino acid analysis, produces a diminution of the free lysine peak and the appearance of a new peak corresponding to epsilon-pyridoxyllysine. The results suggest that there is at least one NH2-lysyl residue of horse muscle acylphosphatase at or near the active site of the enzyme.     

Carbon isotope effects on the decarboxylation of carboxylic acids. Comparison of the lactate oxidase reaction and the degradation of pyruvate by H2O2.

The isotope effect at C-1 on the H2O2-catalysed decarboxylation of pyruvate (used as a model reaction for the enzymic reaction) increases between pH 3 and 10 from 1.0007 +/- 0.0004 to 1.0283 +/- 0.0014 (25 degrees C). This result indicates a change in the rate-determining step from formation of the tetrahedral intermediate to decarboxylation of this intermediate. Practically no isotope fractionation at C-1 (1.0011 +/- 0.0002, pH 6.0, 25 degrees C) is found in the lactate oxidase-catalysed decarboxylation of lactate, which is indicative for the existence of an irreversible O2-dependent step prior to the enzyme-catalysed decarboxylation. In addition, the result provides further evidence that dissociation of pyruvate and H2O2 from the enzyme can be excluded. The isotope effect at C-2 of lactate in the enzymic reaction (1.0048 +/- 0.0004) is attributed to the hydrogen transfer step from lactate to the coenzyme.     

Reversible modification of pig heart mitochondrial malate dehydrogenase by pyridoxal 5''-phosphate.

1. Pig heart mitochondrial malate dehydrogenase incubated with pyridoxal 5'-phosphate at pH 8.0 and 25 degrees C gradually loses activity. Such inactivation can be largely reversed by dialysis or by addition of L-lysine or L-cysteine, and can be made permanent by NaBH4 reduction. 2. Modification of malate dehydrogenase with pyridoxal 5'-phosphate at 35 degrees C involves two phases, an initial inactivation which is reversible and a slower irreversible second stage. 3. The initial reaction between pyridoxal 5'-phosphate and malate dehydrogenase appears to involve reversible formation of a Schiff base with the epsilon-amino group of a lysine residue. 4. Inactivation of malate dehydrogenase by pyridoxal 5'-phosphate at 10 degrees C involves only the reversible reaction. 5. At 10 degrees C repeated cycles of treatment with pyridoxal 5'-phosphate and NaBH4 reduction lead to a stepwise decline in residual activity. 6. Apparent Km values for malate and NAD+ are unaltered in the partially inactivated enzyme. 7. NAD+ and NADH give only partial protection against pyridoxal 5'-phosphate inactivation. Substrates give no effect.     

Active-site-directed inactivation of wheat-germ aspartate transcarbamoylase by pyridoxal 5''-phosphate.

Treatment of 1 microM wheat-germ aspartate transcarbamoylase with 1 mM-pyridoxal 5'-phosphate caused a rapid loss of activity, concomitant with the formation of a Schiff base. Complete loss of activity occurred within 10 min when the Schiff base was reduced with a 100-fold excess of NaBH4. Concomitantly, one amino group per chain was modified. No further residues were modified in the ensuing 30 min. The kinetics of inactivation were examined under conditions where the Schiff base was reduced before assay. Inactivation was apparently first-order. The pseudo-first-order rate constant, kapp., showed a hyperbolic dependence upon the concentration of pyridoxal 5'-phosphate, suggesting that the enzyme first formed a non-covalent complex with the reagent, modification of a lysine then proceeding within this complex. Inactivation of the enzyme by pyridoxal was 20 times slower than that by pyridoxal 5'-phosphate, indicating that the phosphate group was important in forming the initial complex. Partial protection against pyridoxal phosphate was provided by the leading substrate, carbamoyl phosphate, and nearly complete protection was provided by the bisubstrate analogue, N-phosphonoacetyl-L-aspartate, and the ligand-pair carbamoyl phosphate plus succinate. Steady-state kinetic studies, under conditions that minimized inactivation, showed that pyridoxal 5'-phosphate was also a competitive inhibitor with respect to the leading substrate, carbamoyl phosphate. Pyridoxal 5'-phosphate therefore appears to be an active-site-directed reagent. A sample of the enzyme containing one reduced pyridoxyl group per chain was digested with trypsin, and the labelled peptide was isolated and shown to contain a single pyridoxyl-lysine residue. Partial sequencing around the labelled lysine showed little homology with the sequence surrounding lysine-84, an active-centre residue of the catalytic subunit of aspartate transcarbamoylase from Escherichia coli, whose reaction with pyridoxal 5'-phosphate shows many similarities to the results described in the present paper. Arguably the reactive lysine is conserved between the two enzymes whereas the residues immediately surrounding the lysine are not. The same conclusion has been drawn in a comparison of reactive histidine residues in the two enzymes [Cole & Yon (1986) Biochemistry 25, 7168-7174].     

Computational studies on nonenzymatic and enzymatic pyridoxal phosphate catalyzed decarboxylations of 2-aminoisobutyrate

A computational study of nonenzymatic and enzymatic pyridoxal phosphate-catalyzed decarboxylation of 2-aminoisobutyrate (AIB) is presented. Four prototropic isomers of a model aldimine between AIB and 5'-deoxypyridoxal, with acetate interacting with the pyridine nitrogen, were employed in calculations of both gas phase and water model (PM3 and PM3-SM3) decarboxylation reaction paths. Calculations employing the transition state structures obtained for the four isomers allow the demonstration of stereoelectronic effects in transition state stabilization as well as a separation of the contributions of the Schiff base and pyridine ring moieties to this stabilization. The unprotonated Schiff base contribution (approximately 16 kcal/mol) is larger than that of the pyridine ring even when it is protonated (approximately 10 kcal/mol), providing an explanation of the catalytic power of pyruvoyl-dependent amino acid decarboxylases. An active site model of dialkylglycine decarboxylase was constructed and validated, and enzymatic decarboxylation reaction paths were calculated. The reaction coordinate is shown to be complex, with proton transfer from Lys272 to the coenzyme C4' likely simultaneous with C alpha--CO(2)(-) bond cleavage. The proposed concerted decarboxylation/proton-transfer mechanism provides a simple explanation for the observed specificity of this enzyme toward oxidative decarboxylation.     

Stable multisubstrate adducts as enzyme inhibitors. Potent inhibition of ornithine decarboxylase by N-(5'-phosphopyridoxyl)-ornithine.

The synthesis of several N-(5'-phosphopyridoxyl)-amino acids is described. These compounds, analogs of the Schiff base intermediate involved in enzyme-catalyzed decarboxylation, are potent inhibitors of the cognate amino acid decarboxylases. Kinetic studies using partially purified rat liver ornithine decarboxylase, have shown that N-(5'-phosphopyridoxyl)-ornithine inhibits the enzyme in a non-competitive manner with respect to both ornithine and pyridoxal-5'-phosphate. These findings suggest that the inhibitor binds to the holoenzyme active site in place of the Schiff base intermediate.     

Carbon isotope effects on the pyruvate dehydrogenase reaction and their importance for relative carbon-13 depletion in lipids

A method has been developed for the positional 13C isotope analysis of pyruvate and acetate by stepwise quantitative degradation. On its base, the kinetic isotope effects on the pyruvate dehydrogenase reaction (enzymes from Escherichia coli and Saccharomyces cerevisiae) for both of the carbon atoms involved in the bond scission (double isotope effect determination) and on C-3 of pyruvate have been determined. The experimental k12/k13 values with the enzyme from E. coli on C-1 and C-2 of pyruvate are 1.0093 +/- 0.0007 and 1.0213 +/- 0.0017, respectively, and, with the enzyme from S. cerevisiae, the values are 1.0238 +/- 0.0013 and 1.0254 +/- 0.0016, respectively. A secondary isotope effect of 1.0031 +/- 0.0009 on C-3 (CH3-group) was found with both enzymes. The size of the isotope on C-1 indicates that decarboxylation is more rate-determining with the yeast enzyme than with the enzyme from E. coli, although it is not the entirely rate-limiting step in the overall reaction sequence. Assuming appropriate values for the intrinsic isotope effect on the decarboxylation step (k3) and the equilibrium isotope effect on the reversible substrate binding (k1, k2), one can calculate values for the partitioning factor R (k3/k2: E. coli enzyme 4.67, S. cerevisiae enzyme 1.14) and the intrinsic isotope effects related to the carbonyl-C (k1/k'1 = 1.019; k3/k'3 = 1.033). The isotope fractionation at C-2 of pyruvate gives strong evidence that the well known relative carbon-13 depletion in lipids from biological material is mainly caused by the isotope effect on the pyruvate dehydrogenase reaction. In addition, our results indicate an alternating 13C abundance in fatty acids, that has already been verified in some cases.     

Contribution of a conserved arginine near the active site of Escherichia coli D-serine dehydratase to cofactor affinity and catalytic activity

We have employed site-directed mutagenesis to investigate the contribution of a conserved arginyl residue to the catalytic activity and cofactor affinity of D-serine dehydratase, a model pyridoxal 5'-phosphate (vitamin B6) enzyme. Replacement of R-120 in the active site peptide of D-serine dehydratase by L decreased the affinity of the enzyme for pyridoxal 5'-phosphate by 20-fold and reduced turnover by 5-8-fold. kappa cat displayed modified substrate alpha-deuterium isotope effects and altered dependence on both temperature and pH. Analysis of the pH rate profiles of DSD and the R-120----L variant indicated that R-120 interacts electrostatically with catalytically essential ionizable groups at the active site of wild type D-serine dehydratase. The decrease in cofactor affinity observed for DSD(R120L) was not accompanied by significant perturbations in the UV, CD, or 31P NMR spectrum of the holoenzyme, suggesting that the contribution of R-120 to pyridoxal phosphate affinity may be indirect or else involve an interaction with a cofactor functional group other than the 5'-phosphoryl moiety. The properties of two other site-directed variants of D-serine dehydratase indicated that the pyridoxal 5'-phosphate:K-118 Schiff base was indifferent to a small change in the shape of the side chain at position 117 (I-117----L), whereas replacement of K-118 by H resulted in undetectable levels of enzyme. A poor ability to bind cofactor may have rendered DSD(K118H) susceptible to intracellular proteolysis.     

Modification of pyruvate, phosphate dikinase with pyridoxal 5'-phosphate: evidence for a catalytically critical lysine residue

Pyruvate, phosphate dikinase from Bacteroides symbiosus is strongly inhibited by low concentrations of pyridoxal 5'-phosphate. The inactivation follows pseudo-first-order kinetics over an inhibitor concentration range of 0.1-2 mM. The inactivation is highly specific since pyridoxine and pyridoxamine 5'-phosphate, analogues of pyridoxal 5'-phosphate, which lack an aldehyde group, caused little or no inhibition even at high concentrations. The unreduced dikinase-pyridoxal 5'-phosphate complex displays an absorption maxima near 420 nm, typical for Schiff base formation. Following reduction of the Schiff base with sodium borohydride, N6-pyridoxyllysine was identified in the acid hydrolysate. When the enzyme was incubated in the presence of pyridoxal 5'-phosphate and reducing agent, the ATP/AMP, Pi/PPi, and pyruvate/phosphoenolpyruvate isotopic exchange reactions were inhibited to approximately the same extent, suggesting that the modification of the lysyl moiety causes changes in the enzyme that affect the reactivity of the pivotal histidyl residue. Phosphorylation of the histidyl group appears to prevent the inhibitor from attacking the lysine residue. On the other hand, addition of pyridoxal 5'-phosphate to the pyrophosphorylated enzyme promotes release of the pyrophosphate and yields the free enzyme which is subject to inhibition.     

Studies of 6-fluoropyridoxal and 6-fluoropyridoxamine 5'-phosphates in cytosolic aspartate aminotransferase

The chemical and spectroscopic properties of 6-fluoropyridoxal 5'-phosphate, of its Schiff base with valine, and of 6-fluoropyridoxamine 5'-phosphate have been investigated. The modified coenzymes have also been combined with the apo form of cytosolic aspartate aminotransferase, and the properties of the resulting enzymes and of their complexes with substrates and inhibitors have been recorded. Although the presence of the 6-fluoro substituent reduces the basicity of the ring nitrogen over 10 000-fold, the modified coenzymes bind predominately in their dipolar ionic ring forms as do the natural coenzymes. Enzyme containing the modified coenzymes binds substrates and dicarboxylate inhibitors normally and has about 42% of the catalytic activity of the native enzyme. The fluorine nucleus provides a convenient NMR probe that is sensitive to changes in the state of protonation of both the ring nitrogen and the imine or the -OH group of free enzyme and of complexes with substrates or inhibitors. The NMR measurements show that the ring nitrogen of bound 6-fluoropyridoxamine phosphate is protonated at pH 7 or below but becomes deprotonated at high pH around a pKa of 8.2. The bound 6-fluoropyridoxal phosphate, which exists as a Schiff base with a dipolar ionic ring at high pH, becomes protonated with a pKa of approximately 7.1, corresponding to the pKa of approximately 6.4 in the native enzyme. Below this pKa a single 19F resonance is seen, but there are two light absorption bands corresponding to ketoenamine and enolimine tautomers of the Schiff base. The tautomeric ratio is altered markedly upon binding of dicarboxylate inhibitors. From the chemical shift values, we conclude that during the rapid tautomerization a proton is synchronously moved from the ring nitrogen (in the ketoenamine) onto the aspartate-222 carboxylate (in the enolimine). The possible implications for catalysis are discussed.