Pyridoxamine-pyruvate transaminase. I. Determination of the active site stoichiometry and the pH dependence of the dissociation constant for 5'-deoxypyridoxal (original) (raw)
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Kinetics and Mechanism of the Binding of Pyridoxal 5′-Phosphate to Apoglutamate Decarboxylase
Journal of Biological Chemistry, 1972
The rate of binding of pyridoxal 5'-phosphate to apoglutamate decarboxylase from hzherichia coli can be measured by absorption spectroscopy, fluorescence, circular dichroism, and enzyme activity. All four methods give the same results under all conditions examined. At pH 4.9 in pyridinium chloride buffers the binding at a given coenzyme concentration follows tist order kinetics. The rate is not first order in coenzyme, but instead levels off at coenzyme concentrations near 1 mrd. A plot of the reciprocal of the observed first order rate constant uersus the reciprocal of the coenzyme concentration is linear. The binding of 4-deoxypyridoxine 5'-phosphate, like that of pyridoxal 5'-phosphate, follows saturation kinetics, and thus involves at least two distinct steps. The dissociation constant for the first step (0.084 mM) and the rate constant for the second step (0.14 min-I) differ only slightly from those for pyridoxal 5'-phosphate under the same conditions (0.19 mu and 0.20 min-l, respectively). The pK, of the phenolic hydroxyl group of the deoxy compound is shifted downward by more than 3 units on binding to the enzyme. These data are used to derive a general mechanism for the binding of pyridoxal5'-phosphate to glutamate decarboxylase. Coenqme + protein * Corn 1P lex-I Active Enzyme ti Complex-II Complex-I is a rapidly formed complex in which there is weak association between the phosphate of the coenzyme or analog and the protein. The interconversion of Complex-I and Complex-II is a conformation change of the protein and is rate-determining. The catalytically active Schiff base is formed in the last step. The kinetic constants in the above scheme vary according to the anion present. Much faster binding is observed in pyridinium chloride buffer than in pyridinium sulfate or
Biochemistry, 1998
Using a high-performance liquid chromatography-based method for the determination of pyridoxo cofactors, we detected a new intermediate closely related to the inactivation by D-alanine; its formation occurred at the same rate as the inactivation and upon reactivation it reverted to PLP. Conditions were found under which it was characterized by ultraviolet-visible spectral analysis and mass spectroscopy; it is a pyridoxamine phosphate-like compound with a C 2 fragment derived from the substrate attached to the C′-4 of the pyridinium ring and it has a molecular mass of 306 consistent with this structure. In the presence of D-serine, slow accumulation of a quinonoid intermediate is also related to inactivation. The inactivation can be prevented by salts, which possibly stabilize the protonated aldimine coenzyme complex. The reduced cofactor, nicotinamide adenine dinucleotide, prevents D-aspartate-induced inactivation. Both of these events also are related to formation of the novel intermediate. D-Amino acid transaminase catalyzes the transamination of D-amino acids, which are important constituents of the peptidoglycan layer of the bacterial cell wall (1). Transaminases for D-or L-amino acids contain pyridoxal 5′-phosphate (PLP) 1 as cofactor, which is tightly bound both by noncovalent interactions and also covalently to a lysine residue in the active site (2-4). Recently, the crystal structure of D-amino acid transaminase has revealed the various interactions of PLP in the active site (5). The transamination proceeds via a two-step mechanism, in which D-alanine is first bound to the PLP and is converted via a quinonoid intermediate to pyridoxamine phosphate (PMP) with a concomitant release of pyruvate. In the second step, R-ketoglutarate binds to the PMP and, upon formation
2010
We have measured the pH-dependent H, C, and N NMR spectra of pyridoxal 50-phosphate (C2-PLP) mixed with equal amounts of either doubly N-labeled diaminopropane, NR-labeled L-lysine, or Nε-labeled L-lysine as model systems for various intermediates of the transimination reaction in PLPdependent enzymes. At low pH, only the hydrate and aldehyde forms of PLP and the free protonated diamines are present. Above pH 4, the formation of singleand double-headed aldimines (Schiff bases) with the added diamines is observed, and their C and N NMR parameters have been characterized. For 1:1 mixtures the single-headed aldimines dominate. In a similar way, theNMRparameters of the geminal diamine formedwith diaminopropane at high pH are measured. However, no geminal diamine is formed with L-lysine. In contrast to the aldimine formed with the ε-amino group of lysine, the aldimine formed with the R-amino group is unstable at moderately high pH but dominates slightly below pH 10. By analyzing the NMR ...
2008
A study of the reaction between gaseous aldehydes and amines has implicated proton transfer from wall-associated water. Carbinolamine formation and subsequent dehydration to imine with assistance by wall associated hydroxyl bearing species has not previously been specifically suggested to obtain in the multitude of enzyme processes using pyridoxal cofactor. However, the data now available make it clear that imine formation in the active site of those systems requires at least one water or hydroxyl bearing amino acid for proton transfer.
Journal of Biological Chemistry, 2001
Activity of the mammalian pyruvate dehydrogenase complex (PDC) is regulated by phosphorylation-dephosphorylation of three serine residues (designated site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) in the ␣ subunit of the pyruvate dehydrogenase (E1) component. Substitutions of the phosphorylation sites were generated by site-directed mutagenesis. Glutamate (S1E) and aspartate (S1D) substitutions at site 1 resulted in the complete loss of PDC activity; however, these mutants were variably active in the decarboxylation and 2,6-dichlorophenolindophenol assays. S1Q had only 3% of wild-type PDC activity. The apparent K m values for pyruvate increased for the mutants of site 1 when determined in the 2,6dichlorophenolindophenol assay. The substitutions at sites 2 and 3 caused only moderate reductions in activity in the three assays. S3E had a 27-fold increase in the apparent K m for thiamine pyrophosphate and 8-fold increase in the K i for pyrophosphate. Site 3 was almost completely protected from phosphorylation by thiamine pyrophosphate. The results show that the size rather than negative charge of the substituted amino acid residue affects the active site of E1 and that modification of each of the three serine residues affect the active site in a site-specific manner for its ability to bind the cofactor and substrates.
Further studies on the localization of the reactive lysyl residue of pyruvate carboxylase
The Biochemical journal, 1991
We have shown the increase in the acetyl-CoA-independent activity of sheep liver pyruvate carboxylase following trinitrophenylation of a specific lysine residue (designated Lys-A) to be the result of a large stimulation of the first partial reaction and a slight stimulation of the second partial reaction catalysed by this enzyme. Like acetyl-CoA, the activators adenosine 3',5'-bisphosphate and CoA did not stimulate the catalytic activity of the trinitrophenylated enzyme in either the overall reaction or the first partial reaction. Conversely, trinitrophenylation had no effect on activation of the overall reaction and the second partial reaction by acetyl-phosphopantetheine. Protection experiments demonstrated that the presence of both acetyl-CoA and adenosine 3',5'-bisphosphate decreased the rate of loss of activity during exposure of sheep liver pyruvate carboxylase to trinitrobenzenesulphonic acid (TNBS), whereas acetyl-phosphopantetheine did not. 5'-AMP and ac...
Exploring the pyridoxal 5′-phosphate-dependent enzymes
Chemical Record, 2006
Pyridoxal 5′-phosphate (PLP)-dependent enzymes represent about 4% of the enzymes classified by the Enzyme Commission. The versatility of PLP in carrying out a large variety of reactions exploiting the electron sink effect of the pyridine ring, the conformational changes accompanying the chemical steps and stabilizing distinct catalytic intermediates, and the spectral properties of the different coenzyme-substrate derivatives signaling the reaction progress, are some of the features that have attracted our interest to investigate the structure-dynamics-function relationships of PLP-dependent enzymes. To this goal, an integrated approach combining biochemical, biophysical, computational, and molecular biology methods was used. The extensive work carried out on two enzymes, tryptophan synthase and O-acetylserine sulfhydrylase, is presented and discussed as representative of other PLP-dependent enzymes we have investigated. Finally, perspectives of PLPdependent enzymes functional genomics and drug targeting highlight the continuous novelty of an "old" class of enzymes. Scheme 4. Ketoenamine and enolimine tautomers of the external aldimine Schiff base.
Synthesis of pyridoxamine 5′-phosphate using an MBA:pyruvate transaminase as biocatalyst
Journal of Molecular Catalysis B: Enzymatic, 2009
Transaminases (TAs) have useful applications as biocatalysts because of their capability of introducing amino groups into ketones and keto acids with high enantioselectivity, regioselectivity and broad substrate specificity. In this study we have shown that purified His-tagged omega-TA CV2025 from Chromobacterium violaceum is capable of complete conversion of pyridoxal 5 -phosphate (PLP) to pyridoxamine 5 -phosphate (PMP) in the presence of (S)-␣-methylbenzylamine (MBA) as the amine donor. Conversions of 5 mM PLP with at least 0.8 mg/ml CV2025 TA (5.8 U/ml) were complete within 24 h. The fastest completion was achieved with an enzyme concentration of 3 mg/ml (22 U/ml): Within 4 h 5 mM PLP/MBA were converted to 100% and 10 mM PLP/MBA to 70%. PLP amination was only partially inhibited in the presence of 0.5 mM gabaculine, whereas the MBA:pyruvate transamination was shown to be inhibited completely. PMP formation of comparable efficiency could not be achieved with equivalent units of porcine ␣-TA. This represents the first example of a PLP-converting TA with an attributed gene and the first demonstration of quantitative biocatalytic PMP synthesis.
Journal of Biological Chemistry, 1983
The environment of the phosphate group of pyridoxal-P bound at the active site of cytosolic serine hydroxymethyltransferase has been investigated by 3LP NMR spectroscopy. In the holoenzyme, the pyridoxal-P chemical shift is pH-dependent with a p& of 6.45. The chemical shift of the bound pyridoxal-P is shifted upfield about 0.3 ppm from the signal for free pyridoxal-P. Saturation of the active site with the substrates Lserine, glycine, and tetrahydrofolate does not alter the chemical shift or the pK, of the phosphate group. The addition of these substrates does, however, alter the absorption and circular dichroism spectra of the bound coenzyme, reflecting environmental changes of the pyridine ring-Schiff s base system. We conclude from these studies that the phosphate group of the bound coenzyme is exposed to the solvent. The reorientation and conformational changes of the pyridoxal-P ring which take place during the formation of enzyme-substrate complexes do not appear to change the environment of the phosphate moiety of the coenzyme. Cytosolic serine hydroxymethyltransferase (EC 2.1.2.1) catalyzes the formation of glycine and 5,lO-methylenetetrahydrofolate from serine and tetrahydrofolate (1). The enzyme is a