Reactivity and Function of Sulfhydryl Groups in Alanine Dehydrogenase of Bacillus natto KMD 1126

Abstract

Alanine dehydrogenase of Bacillus natto KMD 1126 is composed of 6 identical subunites each with a molecular weight of 48000 daltons. One cysteine residue per subunit of the enzyme was detected by amino acid analysis, and by titration with p-chloromercuribenzoic acid (PCMB) or 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB) in the presence of 8M urea. Titration of the sulfhydryl group with PCMB resulted in a linear decrease in activity with a concomitant increase in the absorbance at 250 nm. The loss of activity was restored by the addition of mercaptoethanol. N-Ethylmaleimide (NEM) inhibited the alanine dehydrogenase with pseudo first order kinetics. One mole of S-succinylcysteine per subunit was detected by amino acid analysis of the NEM inactivated enzyme. Some protection against inactivation by NEM was observed by the addition of nicotinamide adenine dinucleotide (NAD⁺) or its reduced form (NADH). Alanine dehydrogenase was also inhibited by iodoacetamide, and no free sulfhydryl group was detected on titration of the inactivated enzyme with PCMB or DTNB. Gel filtration studies on the binding of NADH to the enzyme revealed that the enzyme contains one coenzyme binding site per subunit.