Abstract
Abstract Rabbit muscle pyruvate kinase is 90% inactivated by binding 2 to 4 moles of pyridoxal 5'-phosphate per mole of tetrameric enzyme. Incubation with pyridoxal-5'-P in 0.2 m imidazole (pH 7.5) results in a time-dependent loss of enzymatic activity which reaches a final value in 10 to 20 min at 25°. Half-maximal loss of activity occurs with 0.04 mm pyridoxal-5'-P. The inactivation is not accompanied by a gross conformational change. The inactivation is first order with respect to pyridoxal-5'-P concentration and enzyme concentration; the second order rate constant is 37 m-1 sec-1. Generally, increasing the ionic strength decreases the rate of inactivation by pyridoxal-5'-P, but low concentrations (1 to 10 mm) of divalent cations increase it above the level with no salt. The phosphate-containing metabolites, ADP, ATP, phosphoenolpyruvate, and fructose diphosphate, also decrease the rate; the effect is more pronounced in the presence of low concentrations (0.1 mm) of divalent cations. The inactivation is relatively specific for pyridoxal-5'-P, since various analogues including pyridoxamine, pyridoxamine 5-phosphate, and pyridoxal cause little or no inactivation. Reduction with NaBH4 at various concentrations of pyridoxal-5'-P shows that there are two types of binding: (a) a specific, inactivating binding, involving 2 to 4 moles of pyridoxal-5'-P bound per mole of enzyme, and (b) a nonspecific, noninactivating binding which can involve 20 or more additional moles of pyridoxal-5'-P bound per mole of enzyme. Both types involve Schiff base formation with e-NH2 groups of lysine. Reversal of the inactivation is accomplished, only with unreduced enzyme, by dilution, by dialysis, or by addition of Tris.
Highlights
0.2 M imidazole results in a time-dependent loss of enzymatic activity which reaches a final value in 10 to 20 min at 25”
The inactivation is not accompanied by a gross conformational change
Increasing the ionic strength decreases the rate of inactivation by pyridoxal-5’-P, but low concentrations (1 to 10 m&r) of divalent cations increase it above the level with no salt
Summary
Rabbit muscle pyruvate kinase was purified and assayed as previously described [15, 17, 18]. The stock enzyme solution (60 mg per ml) was diluted to 3.4 mg per ml with 0.2 M imidazole, pH 7.5, and dialyzed for 10 to 14 hours at 4” against 150 volumes of the buffer. The protein was further diluted to 0.042 mg per ml with solutions containing buffer plus the proper constituents (see “Results”). There was no initial reactivation of the inactive enzyme upon dilution into the assay solution, since the assay was linear for up to 1 min; there was a slow reactivation upon longer incubation since the rate of the reaction increased with time after the first few minutes.
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