Chemical modification of the essential arginine in malate dehydrogenases.

Chemical modification of arginine in malate dehydrogenases from pig heart mitochondria and from Bacillus subtilis was done using 4-hydroxy-3-nitrophenylglyoxal. Incorporation of 2 reagent molecules per subunit was observed concomitantly with complete loss of enzymatic activity. Partial protection was obtained with a substrate analogue and by formation of abortive ternary complexes, whereas coenzyme alone did not inhibit the inactivation. Modified inactive enzymes formed binary complexes with coenzyme as well as the ternary complex with NAD/sulfite. The substrate analogue 8-hydroxy-1,3,6-pyrenetrisulfonate was bound with reduced affinity, however. Because of the known stoichiometry of two reagent molecules per arginine we conclude that one arginine essential for substrate binding was modified in both enzymes.