Magnetic Resonance and Catalytic Studies of Pyruvate Kinase with Essential Sulfhydryl or Lysyl ε-Amino Groups Chemically Modified

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Abstract Rabbit muscle pyruvate kinase has previously been shown to lose its ability to catalyze phosphoryl transfer when an essential lysyl e-amino group in each of the 4 subunits is converted to the N-trinitrophenyl derivative or when an essential sulfhydryl group in each of the 4 subunits is converted to a disulfide. The two modified forms have been examined for their ability to bind Mn2+ and substrates and to catalyze the detritiation of pyruvate, a partial reaction. Both forms bind four Mn2+ ions with an affinity indistinguishable from that of the native enzyme. The disulfide enzyme shows an order of magnitude decrease in the enhanced effect of the bound Mn2+ on the proton relaxation rate of water. Analysis of the temperature and frequency dependencies of the relaxation rates indicates this lowering of enhancement to be due to a decrease in both the number and the exchange rate of rapidly exchangeable water protons on the enzyme-bound Mn2+. The rate of water exchange decreases by two orders of magnitude. The trinitrophenyl-enzyme shows no significant change in the enhancement factor. Ternary enzyme-Mn2+-phosphoenolpyruvate complexes are detected with both modified enzymes. The affinity of the trinitrophenylenzyme-Mn2+ complex for phosphoenolpyruvate is unaltered but the affinity of the disulfide form for phosphoenolpyruvate is decreased by one order of magnitude. No indication of a ternary enzyme-Mn2+-ADP complex is detected with the lysine modification by proton relaxation titrations. With both modifications the ATP-activated detritiation of pyruvate is almost completely inhibited but the Pi-activated detritiation of pyruvate is preserved. With the trinitrophenylenzyme the Km of pyruvate and the Vmax of the detritiation reaction are indistinguishable from those of the native enzyme. With the disulfide modification, however, the Km of pyruvate is increased 3-fold and the Vmax is decreased by 26%. No evidence of ATP binding to the disulfideenzyme is obtained as judged by the effect of ATP on the phosphate-activated detritiation. It is therefore suggested that the sulfhydryl and lysine residues which are modified are both involved in the binding of nucleotides to the enzyme and that the sulfhydryl group is closer to, but not directly at, the Mn2+- and phosphoenolpyruvate-binding sites.