Differential Reactivity of the Thiol Groups of Rabbit Muscle Creatine Kinase

Abstract

Creatine kinase (EC 2.7.3.2) from rabbit muscle is a dimeric enzyme of mol.wt. 82000. The two subunits, which are very similar, if not identical, each possess a reactive thiol group, modification of which by a variety of reagents leads to inactivation of the enzyme (Watts, 1973). A study of the effects of various anions on the properties of the enzyme in the presence of combinations of substrates led to the postulate that a complex resembling the transition state of the reaction can be formed when various small planar anions (e.g. NOaand formate) are added to a mixture of the enzyme with (MgZ++ADP+ creatine) (Milner-White & Watts, 1971). Other studies, using magnetic-resonance techniques, havesupported this view (Reed & Cohn, 1972; James & Cohn, 1974). We have investigated the kinetics of modification of the enzyme by three reagents: 4-chloro-7-nitrobenzofurazan, 5,5’-dithiobis-(2-nitrobenzoic acid) and iodoacetate. Peptide-‘mapping’ experiments had previously shown that iodoacetate reacts with a single thiol group per subunit, (Watts, 1973). The other reagents also react with this thiol group on each subunit as shown by the ability of iodoacetate to protect quantitatively against subsequent reaction with these reagents. The reaction of the enzyme with 4-chloro-7-nitrobenzofurazan in 5OmM-sodium Tricine [N-tris(hydroxymethyI)methylglycine] buffer, pH 7.5 (25”C), followed secondorder kinetics for at least 90% of the total reaction, showing that the two thiol groups (in the dimeric enzyme) reacted at the same rate with the reagent under these conditions. The effect of inclusion of various ligands on the reaction were also studied. ADP and (Mg2++ADP) caused an enhancement in rate; other ligands (e.g. creatine, Mg2+, NO3-) were without significant effect. In all cases, the reactions followed second-order kinetics. In the presence of the ligand combination (MgZ++ADP+creatine+NOa-), two distinct effects were observed. First, the reaction was slowed substantially compared with that performed in the presence of (MgZf+ADP+creatine). Secondly, the second-order kinetic plot showed distinct curvature, indicating that the two thiol groups do not react