Dicarboxylic Substrates Research Articles

A critical arginine residue in cytosolic aspartate aminotransferase from pig heart.

Reaction of the pyridoxal form of cytosolic aspartate aminotransferase from pig heart with 1,2-cyclohexanedione or other alpha-dicarbonyls led to a progressive decrease in the enzymic activity toward natural dicarboxylic substrates. The inactivation was prevented by the presence of dicarboxylic substrate analogs. The dependence of the inactivation rate on the cyclohexanedione concentration indicated that the modifying reagent forms a dissociable complex with the enzyme prior to the inactivation. These saturation kinetics were observed also with other alpha-dicarbonyls tested. The inactivation was fully accounted for by the modification of a single arginine residue per monomeric unit of the enzyme. Activities for alpha, beta-elimination reaction with 3-chloro-L-alanine and transamination with L-alanine did not decrease but appeared to increase considerably with the progress of the arginine modification. In these aberrant reactions, affinity for the monocarboxylic substrates was higher with the modified enzyme than with the native unmodified enzyme. Glutamate or aspartate was still capable of reacting with the pyridoxal form of the extensively modified enzyme to produce the pyridoxamine form at a rate comparable to that of the reaction with 3-chloro-L-alanine or L-alanine. Succinate, glutarate, maleate, 2-methylaspartate or erythro-3-hydroxy-aspartate which bind strongly to the native enzyme and thus acts as potent inhibitors in the reactions with monocarboxylic substrates did not exhibit any appreciable inhibitory effect on these reactions catalyzed by the arginine-modified enzyme. Proton NMR spectroscopy demonstrated that succinate strongly interacts with the native enzyme to generate substantial changes in the enzyme spectra whereas there was no such evidence for the specific interaction with this dicarboxylate with the arginine-modified enzyme.

Involvement of tyrosyl residues in the substrate binding of pigeon liver malic enzyme

The reactions of pigeon liver malic enzyme ( l-malate:NADP + oxidoreductase (oxoloacetate-decarboxylating), EC 1.1.1.40) with tetranitromethane and N- acetylimidazole have been investigated to obtain information about the functional role of tyrosine residues in this enzyme. Incubation of the sulfhydryl-masked enzyme with tetranitromethane or N- acetylimidazole caused a time-dependent loss of all enzymatic activities of this enzyme. The absorption spectra of both the nitrated and acetylated enzyme indicated modification of tyrosine residues. The enzymatic activity of the acetylated enzyme was reversed by hydroxylamine. No amino group modification was observed. Preincubation of the enzyme with dicarboxylate substrate (or inhibitor), nucleotide coenzyme and divalent metal ions protected the enzyme against these reagents. The acetylated enzyme showed different kinetic properties from the native enzyme. The apparent Michaelis constants for malate and oxaloacetate increase by 2–5-fold. The binding between acetylated enzyme and NADPH was not abolished. These results strongly suggest the involvement of tyrosine residues in the dicarboxylic acid binding of malic enzyme.

Mitochondrial and cytosolic aspartate aminotransferase from chicken: Activity toward aromatic amino acids

The mitochondrial and cytosolic isoenzymes of aspartate aminotransferase from chicken heart accept as substrates L-phenylalanine, L-tyrosine and L-tryptophan. The specific activities of the mitochondrial isoenzyme toward these substrates are between 0.1 to 0.5% of that toward aspartate and two orders of magnitude higher than that toward alanine. The specific activities of the cytosolic isoenzyme toward the aromatic substrates are 10 to 70% of the respective values of the mitochondrial isoenzyme. The activities of both isoenzymes toward aromatic amino acids are increased two- to threefold by 1 M formate. Larger increases by formate were observed for the alanine aminotransferase activity of both isoenzymes whereas their aspartate aminotransferase activity was inhibited by formate. The opposite effects of formate on the activities toward the aromatic and aliphatic monocarboxylic substrates on the one hand and the dicarboxylic substrate on the other are consonant with the notion of formate occupying the binding site of the distal carboxylate group of the substrate (Morino Y., Osman A.M., and Okamoto M. (1974) J. Biol. Chem. 249 , 6684–6692). Apparently, in the ternary complex of aspartate aminotransferase with formate and aromatic amino acids, the aromatic rings of the latter bind to a site which does not overlap with the binding site for the distal carboxylate.

Catalytic function of a histidyl residue in cytosolic aspartate aminotransferase-catalyzed reactions

Methylene blue-sensitized photooxidation of the cytosolic aspartate aminotransferase from pig heart yielded a preparation which was inactive in the transamination reaction with natural substrates (L-aspartate and a-keto-glutarate) but fully active in the α, β-elimination reaction with β-chloro-Lalanine as well as in the transamination reaction with L-alanine. The inactivation results from photooxidative destruction of a histidyl residue in accord with the result reported by Martinez-Carrion et al. [ J. Biol. Chem. 242, 1426 (1967)]. These results indicates that the histidyl residue does not function as a base abstracting the α-hydrogen atom of these substrates but acts as a catalytic residue involved in the interaction with the distal carboxylate of a natural dicarboxylic substrate as proposed previously [ Morino et al., J. Biol. Chem. 249, 6684 (1974)].

The binding of 8-anilinonaphthalene-1-sulphonate to cytoplasmic aspartate aminotransferase from pig heart.

Anilinonaphthalenesulphonate binds to cytoplasmic aspartate aminotransferase with high affinity (Kd about 10 muM) and with a stoicheiometry of one molecule per dimer. It is not displaced by aliphatic or aromatic dicarboxylate substrate analogues. The enzyme is believed to be a symmetrical dimer with identical subunits; it can evidently function asymmetrically in binding anilinonaphthalenesulphonate.

Evidence for the presence of a distinct subsite for binding the distal carboxyl group of dicarboxylate substrates and its role in the catalytic activity of aspartate aminotransferase

Rates both of the α,β-elimination of β-chloro-L-alanine and the transamination of L-alanine which are catalyzed by the pyridoxal form of the supernatant isozyme of aspartate aminotransferase were greatly accelerated by the presence of formate. The rate of inactivation of the enzyme occurring during the α,β-elimination reaction was also strikingly enhanced by high concentration of formate. A unified explanation for the formate-mediated rateenhancement of these catalytic processes was attempted by assuming the presence of a discrete subsite within the enzyme active region which binds formate anion and would normally bind the distal carboxyl group of the natural substrate (L-glutamate or L-aspartate). Blocking of this subsite by a carboxylate may induce a conformational change of the catalytic site which favors further chemical reactions in the catalytic process. Thus the present observations provide additional evidence for the active participation of the apparently non-reacting region of a substrate in enzymic catalysis.

Ultraviolet Spectrophotometric Characterization of a Glutamate Dehydrogenase-reduced Coenzyme-α-Ketoglutarate Complex

Abstract Glutamate dehydrogenase binds α-ketoglutarate and TPNH in a ternary complex such that both ligands are more tightly bound than in binary complexes. TPNH, bound in this ternary complex, absorbs maximally at 332 nm with E332 = 5.2 mm-1 cm-1 in contrast to the absorbance of free TPNH (E339 = Emax = 6.2 mm-1 cm-1) and TPNH bound in either a binary or in a glutamate dehydrogenase-TPNH-l-glutamate dead-end ternary complex (E348 = Emax = 4.8 mm-1 cm-1). One molecule of TPNH per 47,000 to 54,000 molecular weight glutamate dehydrogenase (approximately one per peptide chain) was found to be bound in either the α-ketoglutarate or l-glutamate ternary complex. The spectral shifts associated with ternary complex formation were not observed when the coenzyme and substrate analogs, 3-acetylpyridine DPNH or α-ketobutyrate, were used. Therefore, an intact amide on the reduced nicotinamide moiety and a gamma-carboxyl of the dicarboxylate substrate must be required for observable complex formation. The demonstration of the high stability of the glutamate dehydrogenase-TPNH-α-ketoglutarate complex and the correlation of the binding characteristics of the ligands involved in this complex with the effects of these ligands on catalytic events warrants the inclusion of this complex as either a reactive intermediate or a dead-end complex in all general mechanisms proposed for glutamate dehydrogenase catalysis.

Studies of gluconeogenic mitochondrial enzymes: V. The effect of transaminases on reactions catalyzed by glutamate dehydrogenase

Transaminases (the glutamate-oxaloacetate from either bovine liver mitochondria or pig heart and the glutamate-pyruvate from pig heart) are activators of reactions catalyzed by glutamate dehydrogenase. Unlike activation by many ligands as steroids and purine nucleotides, transaminase activates reactions with both monoand dicarboxylic substrates and mainly activates by decreasing the Michaelis constant for ammonium ions. The intact transaminase with pyridoxal phosphate is required for activation. There is much less activation when the concentration of glutamate dehydrogenase is low (reaction with α-ketoglutarate) or in the presence of GTP than when enzyme concentration is high (reaction with pyruvate or oxaloacetate) and in the presence of ADP. Other results are consistent with the concept that glutamate dehydrogenase has transaminase dehydrogenase activity. That is in the presence of TPNH, ammonium chloride and glutamate-oxaloacetate transaminase, glutamate dehydrogenase catalyzes the conversion of the transaminase in the pyridoxal phosphate form to the pyridoxamine phosphate form. Keto-acids can rapidly react with the pyridoxamine form of the transaminase producing the pyridoxal form plus an amino acid. Therefore, in the presence of keto-acids the overall reaction is an amino acid dehydrogenase. This is apparently why transaminases activate reactions catalyzed by glutamate dehydrogenase. The transaminase is not an activator but in the presence of transaminase there are two amino acid dehydrogenase reactions. The transaminase dehydrogenase reaction is significant only in the presence of high concentrations of both enzymes and when α-ketoglutarate and glutamate are either absent or present in low concentrations. It seems that pyridoxal phosphate on the transaminase is bound to the α-ketoglutarate site of glutamate dehydrogenase. Also the transaminase is probably preferentially bound to polymeric forms of glutamate dehydrogenase. Some possible physiological implications of the transaminase dehydrogenase reaction are discussed. This reaction could be of some physiological value when the concentration of amino acids are too low to convert a significant amount of the transaminase into the pyridoxamine form.