O-ALKYL HYDROXAMATES AS METAPHORS OF ENZYME-BOUND ENOLATE INTERMEDIATES IN HYDROXY ACID DEHYDROGENASES - INHIBITORS OF ISOPROPYLMALATE DEHYDROGENASE, ISOCITRATE DEHYDROGENASE, AND TARTRATE DEHYDROGENASE

The inhibition of Thermus thermophilus isopropylmalate dehydrogenase b y O-methyl oxalohydroxamate was studied for comparison to earlier resu lts of Schloss with the Salmonella enzyme. It is a fairly potent (1.2 mu M), slow-binding, uncompetitive inhibitor against isopropylmalate a nd is far superior to an oxamide (25 mM K-i competitive) that is isost eric with the ketoisocaproate product of the enzyme. This improvement in inhibition was attributed to its increased NH acidity, which presum ably is due to the inductive effect of the hydroxylamine oxygen. This principle was extended to the structurally homologous enzyme isocitrat e dehydrogenase from E. coli, for which the compound O-(carboxymethyl) oxalohydroxamate is a 30 nM inhibitor, uncompetitive against isocitra te. The pH dependence of its inhibition supports the idea that it is b ound to the enzyme in the anionic form. Another recently discovered ho mologous enzyme, tartrate dehydrogenase from Pseudomonas putida, was s tudied with oxalylhydroxamate. It has a relatively low affinity for th e enzyme, though it is superior to tartrate. On the basis of these lea ds, squaric hydroxamates with increased acidity compared to squaric am ides directed toward two of these enzymes were prepared, and they also show increased inhibitory potency, though not approaching the nanomol ar levels of the oxalylhydroxamates.