Chemical Modifications of Chicken Liver Pyruvate Carboxylase: Evidence for Essential Cysteine-Lysine Pairs and a Reactive Sulfhydryl Group

Abstract

Inactivations of chicken liver pyruvate carboxylase with N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide (DACM) and o-phthalaldehyde (o-PA) have identified cysteine and lysine residues that are essential for catalytic activity. Protection experiments suggest that the modified residues are located in or near the first and second subsites. At a one- to two-fold molar excess over active site concentration, DACM inactivated approximately 80-90% of the pyruvate carboxylase and ADP/Pi linked oxaloacetate decarboxylase activities by forming a sulfhydryl-DACM adduct with a fluorescence excitation maximum at 385 nm and an emission maximum at 476 nm. o-PA reacted with the enzyme by cross-linking lysine and cysteine residues to form an inactive isoindole-enzyme derivative with a fluorescence excitation maximum at 337 nm and an emission maximum at 415 nm. Incorporation of one equivalent of either DACM or isoindole derivative resulted in an 80-90% decrease in all activities involving chemistry at the first subsite, suggesting that the modification of a sulfhydryl group or a cysteine-lysine ion pair in or near the first subsite inactivates the enzyme. A cysteine-lysine ion pair in the first subsite could function to remove the N-1 proton of biotin to yield enol-biotin, which could be readily carboxylated by the carboxyphosphate intermediate. In the reverse direction, a cysteine-lysine ion pair in or near the second subsite has been proposed to enolize biotin prior to carboxylation by oxaloacetate (P. V. Attwood and W. W. Cleland, 1986, Biochemistry 25,8197-8205). Enzyme modified with 2 equivalents of isoindole retained only 7% of the oxamate-induced, ADP/Pi-independent oxaloacetate decarboxylase activity, suggesting that there is at least one essential cysteine-lysine ion pair at or near the second subsite.