Chemical modifications of chicken liver pyruvate carboxylase: evidence for essential cysteine-lysine pairs and a reactive sulfhydryl group.
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.
Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
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D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002221:Carbamyl Phosphate / Q000378:metabolism; D002645:Chickens; D003545:Cysteine; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D008239:Lysine; D008301:Maleimides / Q000494:pharmacology; D010071:Oxaloacetates / Q000378:metabolism; D010766:Phosphorylation; D011766:Pyruvate Carboxylase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D009764:o-Phthalaldehyde / Q000494:pharmacology
D008301:Maleimides; D010071:Oxaloacetates; D013438:Sulfhydryl Compounds; C019347:N-(7-dimethylamino-4-methylcoumarinyl)maleimide; D002221:Carbamyl Phosphate; D000244:Adenosine Diphosphate; D009764:o-Phthalaldehyde; D011766:Pyruvate Carboxylase; D008239:Lysine; D003545:Cysteine; D005033:Ethylmaleimide