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.     

Ionization state of pyridoxal 5'-phosphate in D-serine dehydratase, dialkylglycine decarboxylase and tyrosine phenol-lyase and the influence of monovalent cations as inferred by 31P NMR spectroscopy

The 31P NMR spectroscopy of three pyridoxal 5'-phosphate-dependent enzymes, monomeric D-serine dehydratase, tetrameric dialkylglycine decarboxylase and tetrameric tyrosine phenol-lyase, whose enzymatic activities are dependent on alkali metal ions, was studied. 31P NMR spectra of the latter two enzymes have never been reported, their 3D-structures, however, are available. The cofactor phosphate chemical shift of all three enzymes changes by approximately 3 ppm as a function of pH, indicating that the phosphate group changes from being monoanionic at low pH to dianionic at high pH. The 31P NMR signal of the phosphate group of pyridoxal 5'-phosphate provides a measure of the active site changes that occur when various alkali metal ions are bound. Structural information is used to assist in the interpretation of the chemical shift changes observed. For D-serine dehydratase, no structural data are available but nevertheless the metal ion arrangement in the PLP binding site can be predicted from 31P NMR data.     

On the coordination of inhibitors to the metal ion of carboxypeptidase A. A 113Cd and 31P NMR study

113Cd and 31P NMR have been used to investigate the interactions of inhibitors with the metal ion of bovine carboxypeptidase A, using 113Cd as a replacement for the native zinc atom. In the absence of inhibitor and over the pH range 6-9, no 113Cd resonance is visible at room temperature. Upon lowering the temperature to 270 K, however, a broad resonance can be seen at 120 ppm. These results are discussed in terms of possible sources for this resonance modulation. Binding of low molecular weight inhibitors containing potential metal-coordinating moieties results in the appearance of a sharp 113Cd resonance. These inhibitors all bind to the metal ion, a fact which is reflected in the chemical shift of the cadmium resonance and, for L-phenylalanine phosphoramidate phenyl ester, by two-bond 113Cd-31P spin-spin coupling of 30 Hz in the 31P resonance of the bound inhibitor. For inhibitors that coordinate to the metal ion via oxygen, the 113Cd chemical shift is in the range 127-137 ppm, whereas for sulfur coordination there is a downfield shift of approximately 210 ppm. The complexes of 113Cd-substituted carboxypeptidase A with the D and L isomers of thiolactic acid are distinguished by a difference of 11 ppm in the chemical shift of their cadmium resonances. The enzyme complex formed with the macromolecular inhibitor from potatoes, which fills the S1 and S2 subsites, shows one or possibly two closely spaced broad 113Cd resonances. Both the chemical shift and the line width of the 113Cd resonances of the [113Cd]carboxypeptidase-inhibitor complexes give valuable structural and dynamic information about the enzyme active site.     

Phosphorus-31 nuclear magnetic resonance of phosphoenzymes of sodium- and potassium-activated and of calcium-activated adenosinetriphosphatase

Prior studies identified phosphoenzyme intermediates in the turnover of sodium- and potassium-activated adenosinetriphosphatase [(Na,K)ATPase] from several sources and of the calcium-activated adenosinetriphosphatase [(Ca)-ATPase] of skeletal muscle sarcoplasmic reticulum. In both cases, the transphosphorylation is to a beta-aspartyl carboxyl group at the active site. We now report observation of a K+-sensitive phosphorylated intermediate of purified (Na,-K)ATPase from the salt gland of the duck using high-field 31P nuclear magnetic resonance. Addition of ATP to a suspension of this enzyme in the presence of Mg2+ and Na+ produced a resonance at about +17 ppm relative to 85% phosphoric acid. Addition of inorganic phosphate and Mg2+ to (Na,K)ATPase also produced a resonance at about +17 ppm which was enhanced in the presence of a saturating concentration of the inhibitor, ouabain; again, addition of K+ made this resonance disappear. These findings are consistent with earlier kinetic characterization of an acid-stable (Na,K)ATPase phosphoenzyme intermediate by 32P-labeled phosphate incorporation into a denatured precipitate of the enzyme. We attribute the +17-ppm resonance to formation of an acyl phosphate at an aspartyl residue of the catalytic site of (Na,K)ATPase. This is supported by our finding of a similar resonance at +17 ppm after phosphorylation of another membrane-bound cation transport enzyme, sarcoplasmic reticulum (Ca)ATPase, as well as by a similar resonance at about +17 ppm after phosphorylation of the model dipeptide L-seryl-L-aspartate.     

Phosphorus-31 nuclear magnetic resonance study of D-serine dehydratase: pryridoxal phosphate binding site.

The pyridoxal phosphate dependent enzyme D-serine dehydratase has been investigated using 31P nuclear magnetic resonance (NMR) at 72.86 MHz. In the native enzyme, the pyridoxal phosphate 31P chemical shift is pH dependent with pKa = 6.4, indicating exposure of the phosphate group to solvent. Binding of the competitive inhibitor isoserine results in the formation of the isoserine-pyridoxal phosphate complex. This transaldimination complex is fixed to the enzyme via the phosphate group of the cofactor as the dianion, independent of pH. At pH 6.6 the dissociation constant KD for isoserine determined by NMR is 0.43 mM. Reconstitution of the apoenzyme with pyridoxal phosphate monomethyl ester produces an inactive enzyme. NMR and fluorescence measurements show that this enzyme does not form the transaldimination complex, indicating that the fixation of the dianionic phosphate (probably via a salt bridge with an arginine residue) observed in the native enzyme is required for the transaldimination step of the catalytic mechanism.     

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.     

Separation and evaluation of the covalent and noncovalent interactions which contribute to the binding of pyridoxal 5'-phosphate to D-serine apodehydratase.

The equilibrium constant (KX) for the reaction D-serine dehydratase + pyridoxamine-P in equilibrium KX D-serine apodehydratase: pyridoxamine-P + pyridoxal-P was determined. At 25 degreees, pH 7.80, KX increases from 5.4 times 10-minus 5 to 21 times 10-minus 5 as T/2 is increased from 0.33 to 0.66. A value of 1.3 times 10-minus 4 M at 25 degrees, pH 7.80, T/2 0.33 for the equilibrium constant (KPMP) for dissociation of pyridoxamine-P from D-serine apodehydratase was determined from the ratio of the equilibrium constant for dissociation of pyridoxal-P from holoenzyme to KX. Pyridoxamine-P and the thiazolidine, formed from pyridoxal-P and cysteine, were found to have similar affinities for D-serine apodehydratase. Using the affinities of these derivatives as a measure of the noncovalent interactions between cofactor and protein, it was possible to estimate the contribution of the Schiff base linkage to the stability of the complex formed between pyridoxal-P and protein. The covalent Schiff base linkage in the holoenzyme was found to be no more stable than the Schiff base linkage formed between 6-aminocaproic acid and pyridoxal-P. The contribution of noncovalent interactions to the stability of the cofactor-protein complex was shown to be at least 20 to 40 times greater than the contribution of the covalent Schiff base linkage.     

A novel zinc-dependent D-serine dehydratase from Saccharomyces cerevisiae

YGL196W of Saccharomyces cerevisiae encodes a putative protein that is unidentified but is predicted to have a motif similar to that of the N-terminal domain of the bacterial alanine racemase. In the present study we found that YGL196W encodes a novel D-serine dehydratase, which belongs to a different protein family from that of the known bacterial enzyme. The yeast D-serine dehydratase purified from recombinant Escherichia coli cells depends on pyridoxal 5'-phosphate and zinc, and catalyses the conversion of D-serine into pyruvate and ammonia with the K(m) and k(cat) values of 0.39 mM and 13.1 s(-1) respectively. D-Threonine and beta-Cl-D-alanine also serve as substrates with catalytic efficiencies which are approx. 3 and 2% of D-serine respectively. L-Serine, L-threonine and beta-Cl-L-alanine are inert as substrates. Atomic absorption analysis revealed that the enzyme contains one zinc atom per enzyme monomer. The enzyme activities toward D-serine and D-threonine were decreased by EDTA treatment and recovered by the addition of Zn2+. Little recovery was observed with Mg2+, Mn2+, Ca2+, Ni2+, Cu2+, K+ or Na+. In contrast, the activity towards beta-Cl-D-alanine was retained after EDTA treatment. These results suggest that zinc is involved in the elimination of the hydroxy group of D-serine and D-threonine. D-Serine dehydratase of S. cerevisiae is probably the first example of a eukaryotic D-serine dehydratase and that of a specifically zinc-dependent pyridoxal enzyme as well.     

Structural basis for VO<Superscript>2+</Superscript> inhibition of nitrogenase activity (A): <Superscript>31</Superscript>P and <Superscript>23</Superscript>Na interactions with the metal at the nucleotide binding site of the nitrogenase Fe protein identified by ENDOR spectroscopy

We previously reported the vanadyl hyperfine couplings of VO(2+)-ATP and VO(2+)-ADP complexes in the presence of the nitrogenase Fe protein from Klebsiella pneumoniae (Petersen et al. in Biochemistry 41:13253-13263, 2002). It was demonstrated that different VO(2+)-nucleotide coordination environments coexist and are distinguishable by electron paramagnetic resonance (EPR) spectroscopy. Here orientation-selective continuous-wave electron-nuclear double resonance (ENDOR) spectra have been investigated especially in the low-radio-frequency range in order to identify superhyperfine interactions with nuclei other than protons. Some of these resonances have been attributed to the presence of a strong interaction with a 31P nucleus although no resolvable superhyperfine structure due to 31P or other nuclei was detected in the EPR spectra. The superhyperfine coupling component is determined to be about 25 MHz. Such a 31P coupling is consistent with an interaction of the metal with phosphorus from a directly, equatorially coordinated nucleotide phosphate group(s). Additionally, novel more prominent 31P ENDOR signals are detected in the low-frequency region. Some of these correspond to a relatively weak 31P coupling. This coupling is present with ATP for all pH forms but is absent with ADP. The ENDOR resonances of these weakly coupled 31P are likely to originate from an interaction of the metal with a nucleotide phosphate group of the nucleoside triphosphate and are attributed to a phosphorus with axial characteristics. Another set of resonances, split about the nuclear Zeeman frequency of 23Na, was detected, suggesting that a monovalent Na+ ion is closely associated with the divalent metal-nucleotide binding site. Na+ replacement by K+ unambiguously confirmed that ENDORs at radio frequencies between 3.0 and 4.5 MHz arise from an interaction with Na+ ions. In contrast to the low-frequency 31P signal, these resonances are present in spectra with both ADP and ATP, and for both low- and neutral-pH forms, although slight differences are detected, showing that these are sensitive to the nucleotide and pH.     

Disruption of active site interactions with pyridoxal 5'-phosphate and substrates by conservative replacements in the glycine-rich loop of Escherichia coli D-serine dehydratase

We have used site-directed mutagenesis to examine the function of three putative active site residues (C278, G279, and G281) of the vitamin B6 enzyme D-serine dehydratase. These residues lie in or adjacent to a conserved glycine-rich loop that is known to interact with the pyridoxal 5'-phosphate cofactor in several B6 enzymes and that resembles the GXGXXG loop of nucleotide-binding sites. The cofactor affinity, catalytic properties, and spectral properties (UV, CD, fluorescence, and 31P NMR) of alanine variants C278A, G279A, and G281A were measured as well as the susceptibility of each variant to thiol modification by 5,5'-dithiobis(2-nitrobenzoic acid). The specific thiols modified in each variant and wild type D-serine dehydratase were identified by amino acid sequencing of labeled tryptic peptides. C278A, G279A, and G281A displayed 10-, 33-, and 22-fold lower affinities for pyridoxal 5'-phosphate than did wild type D-serine dehydratase and turnover numbers with D-serine that were 50, 6, and 60% of normal, respectively. The introduction of a methyl side chain into G281 enhanced catalytic efficiency with the substrates D-threonine, D-allo-threonine, and L-serine, whereas the methyl side chain at position 279 impaired catalysis of all substrates as well as cofactor affinity. The 31P NMR spectrum of D-serine dehydratase was minimally perturbed by the alanine substitutions, consistent with the view that neither G279 nor G281 interacts with the phosphate group of the cofactor (in contrast to the arrangement found in several other B6 enzymes). C311 was the single thiol modified by 5,5'-dithiobis(2-nitrobenzoic acid) in wild type D-serine dehydratase. Two normally inaccessible thiol groups, C233 and C278, were rendered susceptible to modification as a consequence of either G----A substitution, and modification of C278 was associated with inactivation of G279A and G281A. These observations suggest that small perturbations in the glycine-rich loop induce conformational changes spanning a considerable area around the active site.     

The glycine-rich region of Escherichia coli D-serine dehydratase. Altered interactions with pyridoxal 5'-phosphate produced by substitution of aspartic acid for glycine

Replacement of glycine by aspartic acid at either of two sites in a conserved, glycine-rich region inactivates the pyridoxal 5'-phosphate-dependent enzyme D-serine dehydratase (DSD) from Escherichia coli. To investigate why aspartic acid at position 279 or 281 causes a loss of activity, we measured the affinity of the G----D variants for pyridoxal 5'-phosphate and a cofactor:substrate analog complex and compared the UV, CD, and fluorescence properties of wild-type D-serine dehydratase and the inactive variants. The two G----D variants DSD(G279D) and DSD (G281D) displayed marked differences from wild-type D-serine dehydratase and from each other with respect to their affinity for pyridoxal 5'-phosphate and for a pyridoxal 5'-phosphate:glycine Schiff base. Compared to the wild-type enzyme, the cofactor affinity of DSD(G279D) and DSD(G281D) was decreased 225- and 50-fold, respectively, and the ability to retain a cofactor:glycine complex was decreased 765- and 1970-fold. The spectral properties of the inactive variants suggest that they form a Schiff base linkage with pyridoxal 5'-phosphate but do not hold the cofactor in a catalytically competent orientation. Moreover, the amount of cofactor aldamine in equilibrium with cofactor Schiff base is increased in DSD(G279D) and DSD(G281D) relative to that in wild-type DSD. Collectively, our findings indicate that introduction of a carboxymethyl side chain at G-279 or G-281 directly or indirectly disrupts catalytically essential protein-cofactor and protein-substrate interactions and thereby prevents processing of the enzyme bound cofactor:substrate complex. The conserved glycine-rich region is thus either an integral part of the D-serine dehydratase active site or conformationally linked to it.     

Simultaneous bindings of ATP and vanadate to (Na+ + K+)-ATPase. Implications for the reaction mechanism of the enzyme

Inhibition of (Na+ + K+)-dependent adenosine triphosphatase phosphatase by vanadate is thought to occur through the tight binding of vanadate to the same site from which Pi is released. To see if ATP binds to [48V] vanadate-enzyme complex, just as it does to the phosphoenzyme, the effects of Na+, K+, and ATP on the dissociation rate of the complex at 10 degrees C were studied. The rate constant was increased by Na+, and this increase was blocked by K+, indicating that either Na+ or K+ binds to the complex. ATP alone, or in combination with K+, had no effect on the rate constant. In the presence of Na+, however, ATP caused a further increase in the rate constant. The value of K0.5 of Na+ was the same in the presence or absence of ATP; K0.5 of ATP (0.2 mM) did not seem to change significantly when Na+ concentration was varied, and K0.5 of K+, at a constant Na+ concentration, was the same in the presence or absence of ATP. The data indicate that ATP binds to the enzyme-vanadate complex regardless of the presence or absence of Na+ or K+, but it affects the dissociation rate only when Na+ is bound simultaneously. The value of K0.5 of Na+ decreased as pH was increased in the range of 6.5-7.8, but K0.5 of ATP was independent of pH. Demonstration of ATP binding to the enzyme-vanadate complex provides further support for the suggestion that the oligomeric enzyme contains a low-affinity regulatory site for ATP that is distinct from the interacting high-affinity catalytic sites.     

Purification and characterization of recombinant mouse thymidylate synthase

Recombinant mouse thymidylate synthase (TS) expressed at high levels in Escherichia coli was purified to homogeneity in greater than 70% yield by a rapid three-step procedure. Both 0.1% Triton X-100 and 10% glycerol were required to stabilize the enzyme whose activity remained unchanged after 1 month when stored at -20 degrees C. Thermal inactivation of the enzyme was a first-order process at 37 degrees C, with t1/2 values of 6.9, 15.6 and 3.0 min at pH 5.5, 7.0 and 8.5, respectively. The presence of saturating levels of dUMP at pH 8.5 increased the t1/2 of inactivation of 38 min. The pH profile for enzyme activity showed a narrow optimum region centered at pH 7.0, which was mirrored by the shape of the Km, dUMP/Vmax plot. The pH dependence of Kd for the covalent inhibitory ternary complex of enzyme, 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate exhibited a broad minimum between pH 5.5 and 8.5, and ranged between 3.1, 0.8 and 1.1 nM at pH 5.5, 7.0 and 8.5, respectively. The UV/VIS spectrum of the native enzyme exhibited a maximum at 280 nm (epsilon = 98,200 M-1 cm-1), while that of the inhibitory ternary complex showed an additional maximum at 320 nm. The 19F-NMR spectrum of the mouse enzyme:FdUMP binary complex revealed two new resonances at -2.8 and -34.8 ppm. The most deshielded resonance represented the noncovalent binary complex while the other resonance was assigned to the nucleotide covalently bound to the enzyme. The alteration of nucleotide binding equilibria produced by addition of H4 folate was exemplified by both an increase in intensity and a 5 ppm deshielding of the resonance attributed to the covalent FdUMP-enzyme complex. Addition of formaldehyde to the latter mixture produced the covalent ternary complex which resulted in the collapse of the resonances at -2.8 and -39.5 ppm and the appearance of a new resonance at -12.4 ppm.     

D-serine dehydratase from Escherichia coli. DNA sequence and identification of catalytically inactive glycine to aspartic acid variants

We have identified two glycyl residues whose integrity is essential for the catalytic competence of a model pyridoxal 5'-phosphate requiring enzyme, D-serine dehydratase from Escherichia coli. This was accomplished by isolating and sequencing the structural gene from wild type E. coli and from two mutant strains that produce inactive D-serine dehydratase. DNA sequencing indicated the presence of a single glycine to aspartic acid replacement in each variant. The amino acid replacements lie in a glycine-rich region of D-serine dehydratase well removed from pyridoxal 5'-phosphate-binding lysine 118 in the primary structure of the enzyme. The striking effect of these two glycine to aspartic acid replacements on catalytic activity, the conservation of the glycine-rich region in several pyridoxal 5'-phosphate-dependent enzymes that catalyze alpha/beta-eliminations, and the placement of similar glycine-rich sequences in well-characterized active site structures suggest that the glycine-rich region interacts with the cofactor at the active site of the enzyme.     

Bicarbonate- and calcium-dependent induction of rapid guinea pig sperm acrosome reactions by monovalent ionophores

The monovalent cationic ionophores monensin and nigericin stimulated rapid guinea pig sperm acrosome reactions in the presence of extracellular Na+, Ca2+ and bicarbonate (HCO3-/CO2). Extracellular K+ (mM concentrations), in contrast, was not required for the stimulatory effect of the ionophores. The effect of HCO3-/CO2 is concentration, pH and temperature dependent, with maximal responses obtained with 50 microM monensin or 25 microM nigericin at a concentration of 30 mM HCO3-, 2.5% CO2 and pH 7.8 at 25 degrees C. At a constant HCO3- concentration (30 mM), monensin stimulated acrosome reactions within the pH range 7.5-7.8, whereas a higher or lower pH did not support acrosome reactions at 25 degrees C. At constant extracellular pH (7.8), monensin stimulated acrosome reactions in the presence of 30 mM HCO3-, whereas higher and lower concentrations did not support acrosome reactions at 25 degrees C. The permeant anions pyruvate and lactate were essential to maintain sperm motility when treated with monensin under these conditions. NH4Cl, sodium acetate and 4,41-diisothiocyano-2, 21-disulfonic acid stibene (DIDS; 25 microM), an anion transport inhibitor, blocked the ability of monensin to stimulate acrosome reactions. Verapamil (100 microM), a putative Ca2+ transport antagonist, in contrast, did not prevent the monensin-induced acrosome reactions. Physiological concentrations of Na+ were needed for monensin to stimulate acrosome reactions, but high concentrations of Mg2+ prevented the monensin stimulation. The Ca2+ ionophore A23187 (75 nM) also required physiological concentrations of Na+ for the rapid induction of maximal acrosome reactions at an elevated pH (8.3) but did not require the presence of extracellular HCO3-. These studies suggest that a monovalent ionophore-induced rise in sperm intracellular Na+ concentrations is a pre-Ca2+ entry event, that stimulates an endogenous Ca2+/Na+ exchange that allows a Ca2+ influx which in turn induces the acrosome reaction. The possible regulatory role of the sperm intracellular pH and Na+, K+-ATPase during the capacitation process under physiological conditions is discussed.     

Purification and characterization of serine racemase from a hyperthermophilic archaeon, Pyrobaculum islandicum

Pyrobaculum islandicum is an anaerobic hyperthermophilic archaeon that is most active at 100 degrees C. A pyridoxal 5'-phosphate-dependent serine racemase called Srr was purified from the organism. The corresponding srr gene was cloned, and recombinant Srr was purified from Escherichia coli. It showed the highest racemase activity toward L-serine, followed by L-threonine, D-serine, and D-threonine. Like rodent and plant serine racemases, Srr is bifunctional, showing high L-serine/L-threonine dehydratase activity. The sequence of Srr is 87% similar to that of Pyrobaculum aerophilum IlvA (a putative threonine dehydratase) but less than 32% similar to any other serine racemases and threonine dehydratases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration analyses revealed that Srr is a homotrimer of a 44,000-molecular-weight subunit. Both racemase and dehydratase activities were highest at 95 degrees C, while racemization and dehydration were maximum at pH 8.2 and 7.8, respectively. Unlike other, related Ilv enzymes, Srr showed no allosteric properties: neither of these enzymatic activities was affected by either L-amino acids (isoleucine and valine) or most of the metal ions. Only Fe2+ and Cu2+ caused 20 to 30% inhibition and 30 to 40% stimulation of both enzyme activities, respectively. ATP inhibited racemase activity by 10 to 20%. The Km and Vmax values of the racemase activity of Srr for L-serine were 185 mM and 20.1 micromol/min/mg, respectively, while the corresponding values of the dehydratase activity of L-serine were 2.2 mM and 80.4 micromol/min/mg, respectively.     

The role of biotin and sodium in the decarboxylation of oxaloacetate by the membrane-bound oxaloacetate decarboxylase from Klebsiella aerogenes

The biotin-containing oxaloacetate decarboxylase from Klebsiella aerogenes catalyzed the Na+-dependent decarboxylation of oxaloacetate to pyruvate and bicarbonate (or CO2) but not the reversal of this reaction, not even in the presence of an oxaloacetate trapping system. The enzyme catalyzed an avidin-sensitive isotopic exchange between [1-14C]pyruvate and oxaloacetate, which indicated the intermediate formation of a carboxybiotin enzyme. Sodium ions were not required for this partial reaction, but promoted the second partial reaction, the decarboxylation of the carboxybiotin enzyme, thus accounting for the Na+ requirement of the overall reaction. Therefore, the 14CO2-enzyme which was formed upon incubation of the decarboxylase with [4-15C]oxaloacetate, could only be isolated if Na+ ions were excluded. Preincubation of the decarboxylase with avidin also prevented its labelling with 14CO2. The isolated 14CO2-labelled oxaloacetate decarboxylase revealed the following properties. It was slowly decarboxylated at neutral pH and rapidly upon acidification. The 14CO2 residues of the 14CO2-enzyme could be transferred to pyruvate yielding [4-14C]oxaloacetate. In the presence of Na+ this 14CO2 transfer was repressed by the simultaneous decarboxylation of the 14CO2-enzyme. However, Na+ alone was insufficient as a cofactor for the decarboxylation of the isolated 14CO2-enzyme, since this required pyruvate in addition to Na+. It is therefore concluded that the decarboxylation of oxaloacetate proceeds over a CO2-enzyme--pyruvate complex and that free CO2-enzyme is an abortive reaction intermediate. The activation energy of the enzymic decarboxylation of oxaloacetate changed with temperature and was about 113 kJ below 11 degrees C, 60 kJ between 11 degrees C and 31 degrees C and 36 kJ between 31--45 degrees C.     

31P NMR of phosphate and phosphonate complexes of metalloalkaline phosphatases.

31P NMR spectra of phosphate and phosphonate complexes of Escherichia coli alkaline phosphatase have been obtained by Fourier transform NMR methods. One equivalent of P1i, bound to Zn(II) alkaline phosphatase, pH 8, gives rise to a single 31P resonance 2 ppm downfield from that for Pi, and assignable to the noncovalent complex, E-P. Inorganic phosphate in excess of 1 eq per enzyme dimer gives rise to a resonance at the position expected for free Pi. At pH 5.1, a second resonance appears 8.5 ppm downfield from that for free Pi, and is assignable to the covalent complex, E-P. The large downfield shift suggests that the enzyme phosphoryl group is highly strained with an O-P-O bond angle of under 100 degrees.     

113Cd nuclear magnetic resonance of Cd(II) alkaline phosphatases

113Cd NMR spectra of 113Cd(II)-substituted Escherichia coli alkaline phosphatase have been recorded over a range of pH values, levels of metal site occupancy, and states of phosphorylation. Under all conditions resonances attributable to cadmium specifically bound at one or more of the three pairs of metal-binding sites (A, B, and C sites) are detected. By following changes in both the 113Cd and 31P NMR spectra of 113Cd(II)2 alkaline phosphatase during and after phosphorylation, it has been possible to assign the cadmium resonance that occurs between 140 and 170 ppm to Cd(II) bound to the A or catalytic site of the enzyme and the resonance occurring between 51 and 76 ppm to Cd(II) bound to B site, which from x-ray data is located 3.9 A from the A site. The kinetics of phosphorylation show that cadmium migration from the A site of one subunit to the B site of the second subunit follows and is a consequence of phosphate binding, thus precluding the migration as a sufficient explanation for half-of-the-sites reactivity. Rather, there is evidence for subunit-subunit interaction rendering the phosphate binding sites inequivalent. Although one metal ion, at A site, is sufficient for phosphate binding and phosphorylation, the presence of a second metal ion at B site greatly enhances the rate of phosphorylation. In the absence of phosphate, occupation of the lower affinity B and C sites produces exchange broadening of the cadmium resonances. Phosphorylation abolishes this exchange modulation. Magnesium at high concentration broadens the resonances to the point of undetectability. The chemical shift of 113Cd(II) in both A and B sites (but not C site) is different depending on the state of the bound phosphate (whether covalently or noncovalently bound) and gives separate resonances for each form. Care must be taken in attributing the initial distribution of cadmium or phosphate in the reconstituted enzyme to that of the equilibrium species in samples reconstituted from apoenzyme. Both 113Cd NMR and 31P NMR show that some conformational changes consequent to metal ion or phosphate binding require several days before the final equilibrium species is formed.     

31P-nmr studies of the effect of various metals on substrate binding to yeast enolase

The ³¹P nuclear magnetic resonance (nmr) spectra of product (phosphoenolpyruvate) and substrate (2-phosphoglycerate) binding to 1:1 molar ratios ot yeast enolase were obtained as functions of the level of various metal ions. Levels sufficient to produce substrate and product binding but not catalysis ( 1 equivalent/subunit), produced shifts (with respect to 86% H₃PO₄) to lower shielding of ca. 30 ppm in the case of Co²⁺, 5–8 ppm in the case of Mg²⁺, and 2–3 ppm in the case ofCa²⁺, but virtual obliteration in the case of Mn²⁺. The effects of Mn²⁺ and Co²⁺ are consistent with a close approach of the metal ions to the phosphate groups. The effects of the physiological cofactor and optimum activator Mg²⁺ and the nonactivator Ca²⁺ are interpreted as indicating different degrees of distortion of the R-O-P bond angle in the two metal-enzyme-substrate complexes. Levels of Mg²⁺ sufficient for optimal or near optimal catalysis (2 equivalents/subunit) produce shifts to higher shielding in the ³¹P resonances of both substrate and product. These shifts are intermediate between those in the presence of 1 equivalent/subunit and those of the free ligands. Addition of a second equivalent of Ca²⁺ produces a slight shift to lower shielding of the phosphoenolpyruvate resonance and a small shift to higher shielding in the resonance for 2-phosphoglycerate. Similar levels of Co²⁺ eliminate the resonances for both substrate and product. These effects are interpreted as arising from direct coordination between substrate-dependent metal ion binding and the phosphate esters. Higher levels of Ca²⁺, Mg²⁺, or Co²⁺ or addition of KF, all of which inhibit enzyme activity, have only minor effects on the spectra. The spectrum of inorganic phosphate, a competitive inhibitor, was also examined. KF strongly enhances binding, as does excess Mg²⁺, and the binding is accompanied by a chemical shift to lower shielding of ca 2 ppm. This is not due to formation of a magnesium-fluorophosphate complex, consistent with the findings of other workers.