Modification of yeast pyruvate kinase by an active site-directed reagent, bromopyruvate.

The reaction of bromopyruvate with pyruvate kinase was demonstrated both by noting loss of catalytic activity with time and by incorporation of [14C]pyruvate. The first order rate constant for loss of catalytic activity when plotted as a function of bromopyruvate concentration fit a regular hyperbolic saturation function indicative of the formation of a complex with Ki = 15 mM prior to the inactivation event. Twelve moles of [14C]pyruvate were incorporated per mol of protein under these conditions. The alkylation was specific for sulfhydryl residues. When the bromopyruvate reaction was carried out in the presence of substrate, P-enolpyruvate, and modifiers Fru-1,6-Pz, KCl, and MgC12, the reaction with bromopyruvate was second order with Kz = 4.65 M-’ min-l at 30°C. For technical reasons, the number of moles of [14C]pyruvate incorporated/m01 of protein when the reaction mixture contained P-enolpyruvate, Fru-1,6-Pz, KCl, and MgClz could not be determined. Furthermore, P-enolpyruvate, Fru-1,6-Pz, KCl, and MgClz did not protect against inactivation, but rather decreased the rate of inactivation. Using the procedure of Scrutton and Utter (Scrutton, M. C., and Utter, M. F. (1965) J. Biol. Chem. 240,3714-3723), it was possible to show that the ratio of the rate of inactivation determined in the presence of P-enolpyruvate, and modifiers, KCl, MgClz, and Fru-1,6-Pz, to that determined in the absence of modifiers was 0.12. The kinetic properties of the modified enzyme were comparable to the properties of the enzyme modified by 5,5’-dithiobis(2-nitrobenzoic acid) (Yun, S.-l., and Suelter, C. H. (1979) J. Biol. Chem. 254,1806-1810). The sulfhydryl modification studies are consistent with the hypothesis that the sulfhydryl groups are not directly involved in the catalysis, but that one or perhaps two per subunit are located in the vicinity of the catalytic site.