Kinetic mechanism of Escherichia coli isocitrate dehydrogenase and its inhibition by glyoxylate and oxaloacetate.
The inhibition of Escherichia coli isocitrate dehydrogenase by glyoxylate and oxaloacetate was examined. The shapes of the progress curves in the presence of the inhibitors depended on the order of addition of the assay components. When isocitrate dehydrogenase or NADP+ was added last, the rate slowly decreased until a new, inhibited, steady state was obtained. When isocitrate was added last, the initial rate was almost zero, but the rate increased slowly until the same steady-state value was obtained. Glyoxylate and oxaloacetate gave competitive inhibition against isocitrate and uncompetitive inhibition against NADP+. Product-inhibition studies showed that isocitrate dehydrogenase obeys a compulsory-order mechanism, with coenzyme binding first. Glyoxylate and oxaloacetate bind to and dissociate from isocitrate dehydrogenase slowly. These observations can account for the shapes of the progress curves observed in the presence of the inhibitors. Condensation of glyoxylate and oxaloacetate produced an extremely potent inhibitor of isocitrate dehydrogenase. Analysis of the reaction by h.p.l.c. showed that this correlated with the formation of oxalomalate. This compound decomposed spontaneously in assay mixtures, giving 4-hydroxy-2-oxoglutarate, which was a much less potent inhibitor of the enzyme. Oxalomalate inhibited isocitrate dehydrogenase competitively with respect to isocitrate and was a very poor substrate for the enzyme. The data suggest that the inhibition of isocitrate dehydrogenase by glyoxylate and oxaloacetate is not physiologically significant.
D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D006038:Glyoxylates / Q000494:pharmacology; D007521:Isocitrate Dehydrogenase / Q000037:antagonists & inhibitors*; D007700:Kinetics; D009249:NADP / Q000494:pharmacology; D010071:Oxaloacetates / Q000494:pharmacology*
D006038:Glyoxylates; D010071:Oxaloacetates; D009249:NADP; D007521:Isocitrate Dehydrogenase; C031150:glyoxylic acid