Pyruvate-containing enzymes

Decarboxylations of a-amino acids are some of the most widely studied enzymatic reactions, and had been, at one time, presumed to be exclusively associated with pyridoxal-dependent catalysis. The reactivity of an enzyme of this type with carbonyl group reagents such as hydrazines, cyanide or hydroxylamine, was therefore consid- 

Rosenthaler et al. [106] purified histidine decarboxylase from Lactobacillus 30A and demonstrated that there was no pyridoxal phosphate, as had been suggested by Rodwell [107]. Treatment with [ C]phenylhydrazine labeled the protein, but did not if the protein was first reduced with borohydride. Chymotrypsin digestion of the [ C]phenylhydrazone treated enzyme resulted in a labeled fragment identified as A -pyruvoylphenylalanine [100]. Borohydride reduction of the native enzyme resulted in lactate production after hydrolysis. Thus it was established that a pyruvoyl group is covalently bound as an amide to the NH 2-terminal phenylalanine. As is consistent with this proposed mechanism the enzyme is also inhibited by cyanide and by hydroxylamine. The iminium ion predicted by the mechanism above was trapped with borohydride in the presence of substrate and identified [108]. 

The structure of the enzyme appears to be a hexamer, with a dumbell shape 60 A in width and 120 A in length. The native enzyme has a molecular weight of approximately 280000, with a subunit composition and the pro-enzyme has 

Aside from the reduction experiments with or without substrate, and other carbonyl group reactions, evidence has accrued also for the presence of a thiol group at the active site of histidine decarboxylase [12]. It has been demonstrated, for example, that approximately one thiol group per a-fi subunit is titrated with iodoacetamide or p-chloromercuribenzoate, with concomitant inactivation of the enzyme [113]. This same active-site thiol group is titrated with DTNB with loss of activity. Interestingly, the competitive inhibitors histamine and imidazole enhance the reactivity of these thiol residues toward DTNB. Upon denaturation, the enzyme 

The stereochemistry of the overall process has recently been studied. When [S-a- Hjhistidine is used as substrate, the product is [5-a- H]histamine. This observation requires retention of configuration, and is therefore analogous to the stereochemistry of decarboxylations catalyzed by pyridoxal, or by imine formation. 

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Other pyruvate-containing enzymes include aspartate -decarboxylase from Escherichia coli, the enzyme that catalyzes the formation of -alanine for the synthesis of pantothenic acid (Section 12.2.4) proline reductase from Clostridium sticklandiv, phosphatidylserine decarboxylase from E. coli and phenylalanine aminotransferase from Pseudomonas fluorescens. Phospho-pantetheinoyl cysteine decarboxylase, involved in the synthesis of coenzyme A (Section 12.2.1), and S-adenosylmethionine decarboxylase seem to be the only mammalian pyruvoyl enzymes (Snell, 1990). 

CS causes alkylation of sulfhydryl-containing enzymes and inhibits lactic dehydrogenase, glutamic dehydrogenase, pyruvic decarboxy-. lase, and alpha-glycerophosphate dehydrogenase.24,40 it reacts with a number of nucleophilic compounds, such as glutathione, plasma protein, and lipoic acid.24... 

After this, phosphoglycero mutase converts 3-phosphoglycerate into 2-phospho-glycerate, which is then dehydrated in phosphoenol pyruvate by the enzyme eno-lase. Phosphoenol pyruvate contains an energy-rich bond that is used by the enzyme pyruvate kinase to phosphorylate ADP into ATP. This reaction generates pyruvate, which is the final product of glycolysis. 

Pyruvate carboxylase is a well-known Mn metalloenzyme. The enzyme is a tetramer and contains one biotin cofactor per subunit and one divalent cation per subunit. The enzyme from calf liver, for example, contains four tightly boimd Mn atoms. The enzyme from chicken liver contains two Mn atoms and two Mg atoms. Raising chickens on an Mn-deficient diet results in the production of an Mn-free enzyme, where magnesium ions replace the usually occurring manganese ions. The Mg-containing enzyme is catalytically active, leaving the requirement of the enzyme for Mn in question (Scrutton et al, 1972). 

If an asymmetric unit possesses high symmetry, as is often the case for large macromolecu-lar complexes such as the pyruvate dehydrogenase enzyme complex, or icosahedral viruses, then another approach to solving the phase problem becomes available. Viruses, in particular, are amenable because their symmetry operators are very precise and their orientations are well defined. Asymmetric units also occassionally contain redundant copies of a protein that are related by noncrystallographic symmetry through proper or improper rotations and translations. 

N.m.r. has been used to study the active site of other zinc-containing enzymes. In bovine carbonic anhydrase there is a single zinc co-ordination site available for Cl interaction, and the latter can be inhibited by CN and acetazolamide. C1 Resonance has also been used to investigate the environmental diflFerences of the zinc in carbonic anhydrase isozymes and the activation of pyruvate kinase with zinc, and to determine the pJ values in cobalt(u)-carbonic anhydrase. It is suggested that, although the TpK of aquozinc is about 9, the environment of the zinc ion in carbonic anhydrase B greatly increases the tendency of a zinc-bound water molecule... 

This chapter focuses on the microbial fermentation process for lactic acid production. The first commercial operation was set up by Avery in the USA in 1881. Microbes contain enzyme(s) called LDH which can convert pyruvic acid to lactic acid. Depending on the particular microbe and the specificity of its LDH, the lactic acid fermentation process can produce rather pure d-LA or l-LA with high optical purity, or a mixture of them with low optical purity. Genetic engineering tools can be used to knockout the d-LDH gene(s) in the production strain to improve the optical purity of its l-LA fermentation process. 

Another example of a carboxylation reaction is the formation of oxaloacetate from pyruvate. Pyruvate carboxylase (EC 6.4.1.1) consists of 4 subunits, each covalently bound to one molecule of biotin and containing one Mg ion 1. Biotinyl-enzyme + ATP + CO2 + HjO — carboxybiotinyl-enzyme + ADP + Pj 2. Carboxybiotinyl-enzyme + pyruvate biotinyl-enzyme + oxaloacetate. In the degradation of fatty acids with odd numbers of C atoms, carboxylation of pro-pionyl-CoA to methylmalonyl-CoA is also catalysed by B. Carboxybiotinyl-enzyme + CHj-CHj-CO -SCoA biotinyl-enzyme + CHj-CH(COOH)-CO -SCoA. The same reaction occurs in the degradation of isoleucine, leucine and valine. 

Propionyl CoA is carboxylated by a biotin-containing enzyme (C 3.1) to methylmalonyl CoA. Propionic acid is derived from propionyl CoA by transfer of the CoA residue or by hydrolysis. It is degraded via acrylic acid and lactic acid to pyruvic acid (Fig. 83). 

The action of TPP-containing enzymes on pyruvate falls into two groups (1) decarboxylations of the non-oxidative type which lead to products at the aldehyde level of oxidation, namely, acetaldehyde and the acyloins, and (2) decarboxylations of the oxidative type which lead to substances at the acid level of oxidation, such as acetic acid, acetyl phosphate, and acetyl-CoA. 

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