An examination of the role of arginine residues in the functioning of D-glyceraldehyde-3-phosphate dehydrogenase.
Chemical modification of one arginine residue per subunit of tetrameric D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) molecule results in a 85-95% loss of its activity (Nagradova and Asryants (1975) Biochim. Biophys. Acta 386, 365-368; Nagradova, N.K., Asryants, R.A., Benkevich, N.V. and Safronova, M.I. (1976) FEBS Lett. 69, 246-248). Transient kinetic experiments performed in the present work with modified rabbit muscle and Baker's yeast enzymes showed that the first-order rate constant of acyl-enzyme.NADH formation was diminished 30-fold with the rabbit muscle enzyme and 60-fold with the Baker's yeast enzyme. Modification of arginine residues was shown also to affect the second step of the catalytic reaction, the phosphorolysis of the acyl-enzyme (the second-order rate constant of phosphorolysis decreased 9-fold in the case of the rabbit muscle enzyme and 40-fold in the case of the Baker's yeast enzyme). The native and modified enzymes exhibited similar inhibitory constant values with respect to NADH, suggesting no contribution of arginine residues to the acyl-enzyme.NADH complex destabilization. By and large, the experimental data are consistent with the hypothetical scheme proposed on the basis of X-ray crystallography studies to describe a participation of Arg-231 in the catalytic mechanism of D-glyceraldehyde-3-phosphate dehydrogenase (Grau (1982) in the Pyridine Nucleotide Coenzymes, p. 135-187).
Asryants
R A
RA
A.N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, U.S.S.R.
Kuzminskaya
E V
EV
Tishkov
V I
VI
Douzhenkova
I V
IV
Nagradova
N K
NK
D000818:Animals; D001120:Arginine; D001149:Arsenates / Q000494:pharmacology*; D001151:Arsenic / Q000494:pharmacology*; D001665:Binding Sites; D002074:Butanones / Q000494:pharmacology*; D003931:Diacetyl / Q000494:pharmacology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D009132:Muscles / Q000201:enzymology; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D001149:Arsenates; D002074:Butanones; D046911:Macromolecular Substances; C009277:sodium arsenate; D001120:Arginine; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D003931:Diacetyl; D001151:Arsenic