Two-step modification of aspartate aminotransferase with 1,5-difluoro-2,4-dinitrobenzene. Cross-link localization

Rabbit muscle pyruvate kinase has been shown to catalyze the decarboxylation of oxalacetate (Creighton, D.J. and Rose, I.A. (1976) J. Biol. Chem. 251, 61). Noncovalent and covalent modifiers of the enzyme have been used to assess whether the decarboxylase and kinase reactions take place at a common site. Phosphoenol-alpha-ketobutyrate, an analog of the substrate phosphoenol-pyruvate, inhibits decarboxylase and kinase competitively and with nearly identical Ki values (5.7 micron and 4.8 micron, respectively). Oxalate, an analog of enol-pyruvate, inhibits each competitively and with similar Ki values (11 micron and 4.7 micron, respectively). Both activities are lost in parallel upon reaction with dithionitrobenzoate, which is active-site specific. These results indicate that the two activities share a common site on the enzyme. But, effects of the following modifiers suggest that different amino acid residues at that site participate in the two reactions: phenylalanine inhibition and fructose 1,6-bisphosphate activation are more effective with the decarboxylase; iodoacetamide preferentially inactivates decarboxylase while trinitrobenzenesulfonate preferentially inactivates kinase.