The kinetics of Schiff-base formation during reconstitution of D-serine apodehydratase from Escherichia coli with pyridoxal 5'-phosphate.

Schiff base formation during reconstitution of D-serine dehydratase (Escherichia coli) from its apoenzyme and pyridoxal 5'-phosphate (pyridoxal-P) has been studied by rapid kinetic techniques using absorbance changes at 436 nm. Three distinct reaction phases have been observed. The first is a very rapid change during which pyridoxal-P is initially bound to the apoenzyme. This step has an equilibrium constant of 1500 M-1 and a forward reaction rate of the order of 2.6 x 10(6) M-1 s-1. The second phase shows a first-order rate constant with a value dependent on pyridoxal-P and corresponds to a first-order step with a forward rate constant of 3.04 s-1 interacting with the initial equilibrium. The final phase is a slow first-order reaction, the rate constant of which is approximately 0.01 s-1 and is independent of pyridoxal-P concentration. The active pyridoxal species has been shown to be the free pyridoxal-P as opposed to hemiacetal or hemimercaptal forms.     

A spectral probe near the subunit catalytic site of glutamine synthetase from Escherichia coli. Reduced pyridoxal 5'-phosphate.enzyme complexes.

In order to label phosphate binding sites, unadenylylated glutamine synthetase from Escherichia coli has been pyridoxylated by reacting the enzyme with pyridoxal 5'-phosphate followed by reduction of the Schiff base with NaBH4. A complete loss in Mg2+-supported activity is associated with the incorporation of 3 eq of pyridoxal-P/subunit of the dodecamer. At this extent of modification, however, the pyridoxylated enzyme exhibits substantial Mn2+-supported activity (with increased Km values for ATP and ADP). The sites of pyridoxylation appear to have equal affinities for pyridoxal-P and to be at the enzyme surface, freely accessible to solvent. At least one of the three covalently bound pyridoxamine 5'-phosphate groups is near the subunit catalytic site and acts as a spectral probe for the interactions of the manganese.enzyme with substrates. A spectral perturbation of covalently attached pyridoxamine-P groups is caused also by specific divalent cations (Mn2+, Mg2+ or Ca2+) binding at the subunit catalytic site (but not while binding to the subunit high affinity, activating Me2+ site). In addition, the feedback inhibitors, AMP, CTP, L-tryptophan, L-alanine, and carbamyl phosphate, perturb protein-bound pyridoxamine-P groups. The spectral perturbations produced by substrate and inhibitor binding are pH-dependent and different in magnitude and maximum wavelength. Adenylylation sites are not major sites of pyridoxylation.     

Mechanism of action of D-serine dehydratase. Identification of a transient intermediate.

Static absorbance measurements of D-serine dehydratase from Escherichia coli taken at 2 degrees C show that during the steady-state course of D-serine conversion the absorption maximum of the Schiff base of the cofactor pyridoxal 5'-phosphate (pyridoxal-P) is shifted from 415 to 442 nm. Furthermore, the progress curve of intermediates was monitored by stopped-flow techniques at wavelengths ranging from 320 to 500 nm. A point by point construction of successive spectra from these stopped-flow traces at various time intervals after the start of reaction resulted in a series of consecutive spectra exhibiting two isobestic points at 353 and 419 nm. The half-time of the absorbance changes occurring at 330 and 455 nm was found to be 6.5 ms, suggesting the observation of a single, enzyme-bound intermediate. The spectral data with substrate and inhibitors provide evidence that the intermediate is the Schiff base of alpha-aminoacrylate and pyridoxal-P. The proposed assignment is strongly supported by experiments of apodehydratase with transient-state analogues which exhibit a similar absorbance shift on binding to apoenzyme. Moreover, these results suggest that the phosphate group of the substrate--pyridoxal-P complex serves as the main anchoring point during catalysis. A reaction mechanism of the D-serine dehydratase is presented.     

Studies on Vitamin B6 Metabolism in Microorganisms

Oxidation of pyridoxine-P and pyridoxamine-P to pyridoxal-P, inhibition and reactivation of the oxidases were investigated, using the Alcaligenes faecalis oxidase and the Azotobacter agilis oxidase catalyzing. Zone electrophoretic experiments indicated that the oxidases obtained from Alcaligenes faecalis and Azotobacter agilis moved to cathode and anode, respectively, under the same conditions. The oxidation-reduction potential of the both oxidase was found to be about ?50 mV. The oxidation of both pyridoxine-P and pyridoxamine-P was strongly inhibited by pyridoxal-P, pyridoxal, pyridine-4-aldehyde and 4-pyridoxic acid phosphate. This inhibition was markedly decreased by Tris-HCl buffer, and other amino compounds that form Schiff’s base of pyridoxal-P.

An enzyme “pyridoxamine-P transaminase” which catalyzed the transamination between pyridoxamine-P and α-ketoglutaric acid was found in certain anaerobic bacteria, such as Clostridium acetobutylicum, Cl. kainantoi, Cl. kaneboi and Cl. butyricum. The pyridoxamine-P transaminase in the cell-free extract of Cl. kainantoi was purified and some properties were investigated. α-Ketoglutaric acid appeared to be the dominant amino acceptor. Pyridoxamine-P was found to be active as amino donor, but other amino compounds were inert. Since the results were inconclusive, the possibility of vitamin B₆-enzyme of pyridoxamine-P transaminase was not shown by the inhibitor studies. Physiological role of the pyridoxamine-P transaminase was discussed in the relation to vitamin B₆ metabolism in anaerobic bacteria.     

Application of a direct spectrophotometric assay employing a chromogenic substrate for tryptophanase to the determination of pyridoxal and pyridoxamine 5′-phosphates

Improved procedures for the isolation of apotryptophanase and its use in estimation of the vitamin B-6 coenzymes are presented. An excess of the apoenzyme is allowed to react with limiting amounts of pyridoxal-P. Estimation of the holotryptophanase thus formed by use of the chromogenic substrate. S-o-nitrophenyl-l-cysteine, provides a sensitive (1–400 pmol) and conveniently direct spectrophotometric assay for pyridoxal-P. For the specific estimation of pyridoxamine 5′-phosphate, samples are first reduced with NaBH₄ to convert pyridoxal-P to pyridoxine-P (inactive). By nonenzymatic transamination with glyoxylate, pyridoxamine-P is then converted quantitatively to pyridoxal-P and estimated with apotryptophanase. The method gives excellent recoveries of the added coenzymes and indicates that in many tissue extracts pyridoxamine-P surpasses pyridoxal-P in concentration.     

Kinetics and thermodynamics of the interaction of 5-fluoro-2'-deoxyuridylate with thymidylate synthase

Thymidylate synthase (TS), 5-fluorodeoxyuridylate (FdUMP), and 5,10-methylenetetrahydrofolate (CH2-H4folate) form a covalent complex in which a Cys thiol of TS is attached to the 6-position of FdUMP and the one-carbon unit of the cofactor is attached to the 5-position. The kinetics of formation of this covalent complex have been determined at several temperatures by semirapid quench methods. Together with previously reported data the results permit calculation of every rate and equilibrium constant in the interaction. Conversion of the noncovalent ternary complex to the corresponding covalent complex proceeds at a rate of 0.6 s-1 at 25 degrees C, and the dissociation constant for loss of CH2-H4folate from the noncovalent ternary complex is approximately 1 microM. Activation parameters for the formation of the covalent complex were shown to be Ea = 20 kcal/mol, delta G+ = 17.9 kcal/mol, delta H+ = 19.3 kcal/mol, and delta S+ = 0.005 kcal/(mol.deg). The equilibrium constant between the noncovalent and covalent ternary complexes is approximately 2 X 10(4), and the overall dissociation constant of CH2-H4folate from the covalent complex is approximately 10(-11) M. The conversion of the noncovalent ternary complex to the covalent adduct is about 12-fold slower than kcat in the normal enzymic reaction. However, because the dissociation constant for CH2-H4folate from the noncovalent ternary complex is about 10-fold lower than that from the TS-dUMP-CH2-H4folate Michaelis complex, the terms corresponding to kcat/Km are nearly equal. We propose that some of the intrinsic binding energy of CH2-H4folate may be used to facilitate formation of a 5-iminium ion intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of quinolinate on aminotransferases

Quinolinate inhibits several aminotransferases (ornithine, alanine, and aspartate). However, it is considerably more potent as an inhibitor of liver and heart cytoplasmic aspartate aminotransferase. It is a much less potent inhibitor of mitochondrial aspartate aminotransferases. Quinolinate is bound to the active site of cytoplasmic aspartate aminotransferase. It has a much greater affinity for the pyridoximine-P than the pyridoxal-P form of the enzyme. According to kinetic results, the inhibition or dissociation constant of quinolinate is 0.2 and 20 mm, respectively, for the pyridoxamine-P and the pyridoxal-P forms of the enzyme. Since quinolinate is mainly bound to the pyridoxamine-P form: (a) it is a potent competitive inhibitor of α-ketoglutarate but has little effect when α-ketoglutarate is saturating even if the level of aspartate is low; (b) it decreases the effect of α-ketoglutarate on the absorption spectrum of the pyridoxamine-P form; and (c) it enhances the effect of glutamate on the absorption spectrum of the pyridoxal-P form. Quinolinate is also apparently bound to the apoenzyme since it inhibits reconstitution by either pyridoxamine-P or pyridoxal-P. Since quinolinate is a competitive inhibitor of α-ketoglutarate, it is possible that part of the inhibitory effect of quinolinate on hepatic gluconeogenesis could result from quinolinate inhibiting the conversion of aspartate to oxalacetate by the cytoplasmic aspartate aminotransferase. Quinolinate has no effect on either rat or bovine liver glutamate dehydrogenase or on kidney glutamate dehydrogenase.     

Characterization of the pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate hydrolase activity in rat liver. Identity with alkaline phosphatase.

The tissue content of pyridoxal 5'-phosphate is controlled principally by the protein binding of this coenzyme and its hydrolysis by a cellular phosphatase. The present study identifies this enzyme and its intracellular location in rat liver. Pyridoxal-P is not hydrolyzed by the acid phosphatase of intact lysosomes. At pH 7.4 and 9.0, the subcellular distribution of pyridoxal-P phosphatase activity is similar to the for p-nitrophenyl-P, and the major portion of both activities is found in the plasma membrane fraction. The ratio of specific activities for pyridoxal-P and p-nitrophenyl-P hydrolysis remains relatively constant during the isolation of plasma membranes. These activities also behave concordantly with respect to pH rate profile, pH-Km profile, and response to chelating agents, Zn2+, Mg2+, and inhibitors. Kinetic studies indicate that pyridoxal-P binds to same enzyme sites as beta-glycerophosphate and phosphorylcholine. The data strongly favor alkaline phosphatase as the enzyme which functions in the control of pyridoxal-P and pyridoxamine-P metabolism in rat liver. Alkaline phosphatase was solubilized from isolated plasma membranes. The kinetic properties of the enzyme are not markedly altered by its dissociation from the membrane matrix. However, there are significant differences in its behavior toward Mg2+ which suggest a structural role for Mg2+ in liver alkaline phosphatase.     

The antibody-enzyme analogy. Comparison of enzymes and antibodies specific for phosphopyridoxyltyrosine.

Reduced Schiff base compounds of pyridoxal-P and tyrosine, which were used to induce specific antibodies described in the preceding article (V. Raso and B. D. Stolar, Biochemistry, 1975), caused active site-directed inhibition of tyrosine transaminase and tyrosine decarboxylase. The antibodies, studied as analogs of enzymes, were able to bind an unsaturated Schiff base catalytic intermediate, as shown by equilibrium dialysis and absorbance difference spectroscopy. Schiff base formation can proceed while the pyridoxal-P and tyrosine are within the antibody combining site, but the rate of this bimolecular condensation within the sites was not greater than the rate in free solution. Antibody did effect a small rate enhancement for the pyridoxal-P-catalyzed transamination of L-tyrosine. These results are discussed in light of current ideas in the mechanisms of enzyme catalysis.     

Structure and function of D-amino acid oxidase. IX. Changes in the fluorescence polarization of FAD upon complex formation.

1. The fluorescence polarization, P, of FAD increased on complex formation with the apoenzyme of D-amino acid oxidase [D-amino acid: O2 ocidoreductase (deaminating), EC 1.4.3.3]. The time course of the increase was monophasic. The values of P were extimated to be 0.04, 0.4, and 0.4 for FAD, the enzyme and the enzyme-benzoate complex, respectively. 2. The value of P of the enzyme is dependent on its concentration, indicating that the degrees of dissociation of FAD in the monomer and dimer are different. The dissociation constant was calculated to be 7 times 10-minus 7 M for the monomeric form of the enzyme. This value is far larger than the value for the dimeric form of the enzyme, 1 times 10-minus 8 M, calculated from equilibrium dialysis data. 3. Changes in fluorescence polarization of the enzyme due to changes in solution pH or temperature can be explained in terms of the monomer-dimer equilibrium.     

The role of Lys272 in the pyridoxal 5-phosphate active site of Synechococcus glutamate-1-semialdehyde aminotransferase.

Glutamate-1-semialdehyde (GSA) aminotransferase catalyzes transfer of the C2 amino group of glutamate 1-semialdehyde to the C1 position to yield the tetrapyrrole precursor 5-aminolevulinate. Based on spectrophotometric and steady-state data, GSA aminotransferase is a B6-containing enzyme which uses a ping-pong bi-bi mechanism described for other aminotransferases. A putative active-site lysine at position 272 of Synechococcus GSA aminotransferase was replaced by Arg, Ile or Glu, and genes encoding the corresponding three site directed mutants were expressed in Escherichia coli. The catalytic competence of the resulting enzymes was determined. The similarity of the absorbance spectra of pyridoxal-P-treated forms of Lys272----Arg, Lys272----Ile, Lys272----Glu with free pyridoxal-P indicates that enzyme-bound pyridoxal-P does not form an internal aldimine in in these three site-directed mutants. Whereas Lys----Ile and Lys----Glu form only stable ketimines and aldimines with GSA and its analogues, addition of these compounds to the pyridoxamine-P and pyridoxal-P forms of Lys----Arg induces slow spectral changes, indicating the catalysis of a half-reaction with GSA, 4,5-dioxovalerate and 4,5-diaminovalerate. 5-Aminolevulinate apparently binds with both coenzyme forms of Lys272----Arg, however significant tautomeric rearrangement is only observed with the pyridoxal-P form. It is suggested that Lys272 is the covalent pyridoxal-P-binding site and that this catalytically active lysine residue channels the overall transamination reaction towards 5-aminolevulinate. The second-half reaction (4,5-diaminovalerate in equilibrium with 5-aminolevulinate) is possibly supported by the formation of an internal aldimine which correctly positions the C4 amino group of 4,5-diaminovalerate relative to the enzyme-bound pyridoxal-P.     

Covalent and noncovalent receptor-glucocorticoid complexes preferentially bind to the same regions of the long terminal repeat of murine mammary tumor virus proviral DNA

Dexamethasone 21-mesylate, an irreversible antiglucocorticoid in HTC cells, forms a covalent receptor-steroid complex which can be activated in cell-free systems. The molecular basis of its antiglucocorticoid activity is unknown; it might result from altered DNA sequence preferences and/or affinities of the covalent receptor-steroid complex. To test this hypothesis, the affinities of both covalent receptor-antagonist and noncovalent receptor-agonist complexes for defined DNA sequences were measured in a DNA binding competition assay. This assay requires neither purified complexes nor large quantities of DNA, yet it provides quantitative comparisons of the affinities of different double-stranded DNAs for binding receptor-steroid complexes. In this assay, activated covalent receptor-dexamethasone 21-mesylate complexes in crude cytosol bound to calf thymus DNA and cloned subregions of the long terminal repeat (LTR) of murine mammary tumor virus (MMTV) proviral DNA with approximately the same relative affinities as did noncovalent receptor-dexamethasone complexes. Both types of complex exhibited similar orders of preferential binding to DNA sequences. LTR subregions, as well as the entire LTR, were 2-20 times more potent competitors than calf thymus DNA. Cloned sequences from the 3' terminus of the LTR were more effective competitors than either the entire LTR or comparably sized DNAs from the 5' terminus. The DNA sequences with the greatest affinities for both covalent and noncovalent complexes are located within the region of -221 to -67. These studies support the theory that recognition by regulatory elements of specific DNA sequences upstream of responsive genes is an integral step of hormone action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of the glycation of bovine serum albumin by mannose and fucose in vitro

Glycation of bovine serum albumin was measured for mannose and fucose at 37 degrees C. Mannose as well as fucose demonstrated an initial rapid increase in rate of formation of total adducts followed by a slower secondary reaction. The equilibrium constant for Schiff base formation was almost two times larger for mannose than fucose, although the Schiff base formed by fucose rearranged 1.5 times faster than that for mannose. Both sugars showed parallel lines for the formation of total and acid stable products after three hours. Discussion integrates new mechanistic data with previously suggested mechanisms.     

Substrate-induced changes in sulfhydryl reactivity of bacterial D-amino acid transaminase

D-Amino acid transaminase from Bacillus sphaericus strain ATCC 14577 is a dimer with eight cysteinyl residues per molecule (T.S. Soper, W.M. Jones, and J.M. Manning (1979) J. Biol. Chem. 254, 10,901-10,905). The reaction of the cysteinyl residues with a variety of sulfhydryl reagents has been explored to gain insight into the physical environments around these cysteinyl residues in the absence or the presence of substrates. The native enzyme, in the pyridoxal-P conformation, appears to be a symmetrical dimer, whose SH groups react in pairs with anionic reagents such as 5,5'-dithiobis(2-nitrobenzoic acid) or the halo acids. Two SH groups react with either reagent without altering enzymatic activity. Two additional SH groups react with DTNB with loss of catalytic activity. Positively charged reagents such as beta-bromoethylamine are much more effective in inactivating the pyridoxal-P conformation of the enzyme with almost five of the eight SH groups reacting and this results in a significant loss in catalytic activity. The neutral reagent dithiodipyridine is able to detect some asymmetry in the pyridoxal-P conformation. Upon addition of a D-amino acid substrate, the enzyme is transformed into the pyridoxamine-P conformation. This conformation is much more reactive with anionic reagents and much less reactive with cationic reagents, suggesting that there is a significant change in the net charge around one of the SH groups in the pyridoxamine-P conformation. Also, titration with DTNB indicates that the enzyme is a much more asymmetric dimmer in the pyridoxamine-P conformation than in the pyridoxal-P conformation. Thus, upon binding of a D-amino acid substrate, D-amino acid transaminase is transformed into the pyridoxamine-P conformation. This results in a significant change in the environment of four of the sulfhydryl groups of the enzyme. We conclude that the enzyme is transformed from a symmetrical dimer into an asymmetrical dimer and that the net charge of one of the pairs of cysteinyl groups is changed from a net negative charge into a net positive charge. These results suggest that there is a significant conformational change that occurs during the transition from the pyridoxal-P into the pyridoxamine-P form of this transaminase.     

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.     

Binding of colchicine to purified microtubule protein.

The binding of colchicine to tubulin, purified by two cycles of assembly-disassembly, has been studied. Equilibrium studies indicated a dissociation constant which declined during incubation approaching a minimum value of approximately 0.30 times 10- minus 6 M after 13 hours of incubation. Because tubulin is unstable during prolonged incubation (t1/2 of 5.2 hours for free tubulin, t1/2 of 12.5 hours for tubulin bound to colchicine), the equilibrium Kd was felt to be an overestimation of the true Kd. The rate constant of dissociation (k-1 equal to 0.009 hour- minus 1 hour- minus 1) and the rate constant of association (k1 equal to 0.37 times 10-6 M-minus 1) were measured under conditions designed to circumvent or correct for tubulin instability. The dissociation constant determined by the ratio k-1/k1 was 0.024 times -minus 6 M. To determine whether the discrepancy between the "equilibrium" and "kinetic" determined dissociation constants could be accounted for on the basis of tubulin instability, the binding reaction was computer-simulated using the measured association and dissociation rate constants and the rate constants for decay of bound and free tubulin. Computer simulation was in close agreement with the experimentally determined behavior of the reaction during a 13-hour incubation. It is concluded that the Kd determined by equilibrium methodology results in a considerable overestimation due to the instability of tubulin, and that the best estimate for the Kd of the colchicine-tubulin equilibrium is the value determined by the ratio of the rate constants.     

Multiwavelength analysis of the aldehyde hydration of pyridoxal phosphate: A stopped flow study

A simple method has been devised to determine the rate constants of the aldehyde hydration of pyridoxal-P by coupling the hydration reaction to the formation of a Schiff base derived from free pyridoxal-P and a primary amine present in excess. The primary amine component to be chosen should have a pK value very close to the pH at which the hydration ratio is to be determined. At pH 7.8 glycine ethyl ester is particularly well suited as primary amine component due to its favorable pK of 7.83. The overall reaction consisting of two independent reaction steps was monitored at different wavelengths and analyzed by means of the formal integration method. The rate constant of the forward reaction (k_h¹ = k_h [H₂O]) and of the reverse reaction (k_d) were found to be 1.99 s^?1 and 8.09 s^?1, respectively. The corresponding equilibrium constant is 0.25.     

Diaminopropionate ammonia-lyase from Salmonella typhimurium. Purification and characterization of the crystalline enzyme, and sequence determination of the pyridoxal 5'-phosphate binding peptide

We have found a wide occurrence of alpha,beta-diaminopropionate ammonia-lyase in bacteria and actinomycetes. Considerable amounts of this enzyme were found in Salmonella typhimurium. The enzyme was purified and crystallized from S. typhimurium (IFO 12529). The relative molecular mass of the native enzyme, estimated by the ultracentrifugal equilibrium method, is 89,000 Da, and the enzyme consists of two subunits identical in molecular mass. The enzyme exhibits absorption maxima at 278 and 413 nm and contains 2 mol of pyridoxal 5'-phosphate(pyridoxal-P)/mol of enzyme. The enzyme catalyzes the alpha,beta-elimination reaction of both L- and D-alpha,beta-diaminopropionate, the most suitable substrates, to form pyruvate and ammonia. The L- and D-isomers of serine were also degraded, though slowly. After the internal Schiff base with pyridoxal-P had been reduced with sodium borohydride, followed by trypsin or lysyl endopeptidase digestion of the enzyme, we determined the sequence of about 20 amino acid residues around the lysine residue which binds pyridoxal-P. No homology was found in either the amino acid sequence of the pyridoxal-P binding peptide or the amino-terminal amino acid sequence between the enzyme and other pyridoxal-P-dependent enzymes.     

Purification and characterization of vitamin B6-phosphate phosphatase from human erythrocytes.

Human erythrocytes rapidly convert vitamin B6 to pyridoxal-P and contain soluble phosphatase activity which dephosphorylates pyridoxal-P at a pH optimum of 6-6.5. This phosphatase was purified 51,000-fold with a yield of 39% by ammonium sulfate precipitation and chromatography on DEAE-Sepharose, Sephacryl S-200, hydroxylapatite, and reactive yellow 86-agarose. Sephacryl S-200 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme was a dimer with a molecular mass of approximately 64 kDa. The phosphatase required Mg2+ for activity. It specifically catalyzed the removal of phosphate from pyridoxal-P, pyridoxine-P, pyridoxamine-P, 4-pyridoxic acid-P, and 4-deoxypyridoxine-P at pH 7.4. Nucleotide phosphates, phosphoamino acids, and other phosphorylated compounds were not hydrolyzed significantly nor were they effective inhibitors of the enzyme. The phosphatase showed Michaelis-Menten kinetics with its substrates. It had a Km of 1.5 microM and a Vmax of 3.2 mumol/min/mg with pyridoxal-P. The Vmax/Km was greatest with pyridoxal-P greater than 4-pyridoxic acid-P greater than pyridoxine-P greater than pyridoxamine-P. The phosphatase was competitively inhibited by the product, inorganic phosphate, with a Ki of 0.8 mM, and weakly inhibited by pyridoxal. It was also inhibited by Zn2+, fluoride, molybdate, and EDTA, but was not inhibited by levamisole, L-phenylalanine, or L(+)-tartrate. These properties of the purified enzyme suggest that it is a unique acid phosphatase that specifically dephosphorylates vitamin B6-phosphates.     

Decarboxylation-dependent transamination catalyzed by mammalian 3,4-dihydroxyphenylalanine decarboxylase

In addition to the usual decarboxylation, pig kidney 3,4-dihydroxyphenylalanine (dopa) decarboxylase catalyzes a decarboxylation-dependent transamination which converts dopa into 3,4-dihydroxyphenylacetaldehyde and sinultaneously converts enzyme-bound pyridoxal-P into pyridoxamine-P. Similar reactions occur when this enzyme acts on m-tyrosine, alpha-methyldopa, and alpha-methyl-m-tyrosine. The transamination occurs in about 0.02% of decarboxylations of dopa and m-tyrosine and in about 2% of decarboxylations of alpha-methyldopa and alpha-methyl-m-tyrosine. The fraction of decarboxylations proceeding by the transamination pathway is independent of pH. This reaction appears to result from a divergence in the normal mechanism of decarboxylation; the quinoid intermediate which is formed by decarboxylation of the substrate-pyridoxal-P-Schiff base ordinarily protonates on the alpha carbon of the amino acid, but protonation occasionally occurs at the benzylic carbon of the coenzyme, and this latter route leads to transamination.