Identification of a Cysteinyl Residue Involved in the Activity of Rabbit Muscle Aldolase

Abstract

We have previously shown in 1969 that three out of seven cysteinyl residues per subunit can be selectively carboxymethylate in rabbit muscle aldolase with bromoacetate with the concomitant loss of enzymic activity. Kinetic analysis has shown that inactivation was due to the blocking of the second SH-group (Cys-II) in the order of decreasing reactivity towards the reagent. In the present work we present evidence that Pi protects Cys-II from carboxymethylation whereas it has no effect on the alkylation of Cys-I. The partial sequence analysis of the tryptic peptides containing Cys-I, Cys-II, and Cys-III is also presented. Heat denaturation studies indicate that the alkylated enzyme is less stable than the native one and that alkylation damages the phosphate binding sites of aldolase. The apparent dissociation constant of the tight enzyme-phosphate complex is increased by one order of magnitude, and the loose enzyme-phosphate complex is rendered undetectable.