Studies on regulatory functions of malic enzymes. IV. Effects of sulfhydryl group modification on the catalytic function of NAD-linked malic enzyme from Escherichia coli.
Reactions catalyzed by NAD-linked malic enzyme from Escherichia coli were investigated. In addition to L-malate oxidative decarboxylase activity (Activity 1) and oxaloacetate decarboxylase activity (Activity 2), the enzyme exhibited oxaloacetate reductase activity (Activity 3) and pyruvate reductase activity (Activity 4). Optimum pH's for Activities 3 and 4 were 4.0 and 5.0, and their specific activities were 1.7 and 0.07, respectively. Upon reaction with N-ethylmaleimide (NEM), Activity 1 decreased following pseudo-first order kinetics. Activity 2 decreased in parallel with Activity 1, while Activities 3 and 4 were about ten-fold enhanced by NEM modification. Modification of one or two sulfhydryl groups per enzyme subunit caused an alteration of the activities. Tartronate, a substrate analog, NAD+, and Mn2+ protected the enzyme against the modification. The Km values for the substrates and coenzymes were not significantly affected by NEM modification. Similarly, other sulfhydryl reagents such as p-hydroxymercuribenzoate (PMB), 5,5'-dithiobis(2-nitrobenzoate) (DTNB), and iodoacetate inhibited the decarboxylase activities and activated the reductase activities to various extents. Modification of the enzyme with PMB or DTNB was reversed by the addition of a sulfhydryl compound such as dithiothreitol or 2-mercaptoethanol. Based on the above results, the mechanism of the alteration of enzyme activities by sulfhydryl group modification is discussed.
D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000378:metabolism*; D009243:NAD; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D009243:NAD; D004228:Dithionitrobenzoic Acid; D008291:Malate Dehydrogenase; D005033:Ethylmaleimide