Proton NMR studies of the histidine residues of rabbit muscle pyruvate kinase and of its phosphoenolpyruvate complex.

Abstract

Proton NMR spectra at 250 MHz of rabbit muscle pyruvate kinase, a tetrameric protein of molecular weight 237,000, were obtained with 16 bit A/D conversion in order to study the roles of the histidine residues in catalysis. Six of the 14 histidine residues/subunit gave detectable C-2 proton signals, and three gave C-4 proton signals. Analysis of the upfield shifts of these resonances with increasing pH by least squares computer fitting indicated these signals to arise from single histidines with the following pK' values at 25 C in the presence of the activating monovalent (K+) and divalent (Mg?+) cations: His-1,6.7; His-2,6.3; His-3,6.2; His4, 6.0; His-5, 5.9; and His-6, 5.6. The presence of a saturating level of the substrate P-enolpyruvate resulted in a 0.4 unit decrease in the pK' of His-3 to a value of 5.8, but no significant change in any of the other pK' values. Titration with P-enolpyruvate at a constant pH* (5.8) measuring the upfield shift of the His-3 resonance yielded a dissociation constant for Penolpyruvate (74 2 7 a~) consistent with kinetic and other binding data, indicating active site binding. Both the monovalent (K+) and divalent (M&+) cation activators were found to be essential for the full P-enolpyruvate induced upfield shift. No effects of P-enolpyruvate were noted in the absence of both cations and only small effects of P-enolpyruvate were noted in the presence of either cation activator alone. The metal activators themselves induced smaller upfield shifts of the His-3 resonance. These findings indicate that the decrease in pK' of His-3 induced by the binding of Penolpyruvate results directly from a stronger interaction of the metal ion activators with His-3, in the enzyme-substrate complex, or indirectly from a P-enolpyruvate induced conformational change in the environment of His-3.