CHEMICAL MODIFICATION OF ARGINYL RESIDUES IN RABBIT MUSCLE PYRUVATE-KINASE
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1981
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Abstract
Rabbit muscle pyruvate kinase was inactivated with the arginine-specific reagent 2,3-butanedione, in borate buffer, following psuedo-first-order kinetics. Graphical representation of the kobs as a function of inactivator concentration follows a straight line which is compatible with the following mechanism: .**GRAPHIC**. Values for k1 = 4.6 M-1 min-1 and k1 = 0.0022 min-1 can be estimated. The order of the reaction was near 1, suggesting that the modification of a single amino acid residue of the enzyme is responsible for its inactivation. Almost full catalytic activity was recovered when excess butanedione and borate buffer were removed by Sephadex G-25 gel filtration, which suggests that only arginyl residues were modified. The inactivation was partially prevented by phosphoenolypyruvate in the presence of K+ and Mg2+, but not by the competitive inhibitors lactate and bicarbonate, or ADP, with or without added metal ions. Mg2+ alone increased the rate of inactivation. Quantification of residual arginine residues after chemical modification, using amino acid analysis, gives no precise correlation between the number of residues modified and the residual activity; however, .apprx. 4 residues react per enzyme subunit, showing the presence of reactive, nonessential arginines. One essential arginyl residue is present per subunit in rabbit muscle pyruvate kinase, and this residue is probably located near the binding site of the phosphate group of phosphoenolpyruvate.