Effects of temperature and reagent size on the reaction of the thiol groups of rabbit muscle creatine kinase [proceedings].

The dimeric enzyme creatine kinase (EC 2.7.3.2) from rabbit muscle has a reactive thiol group on each subunit, modification of which by iodoacetate or iodoacetamide leads to inactivation of the enzyme (Watts, 1973). It had been previously shown (Price & Hunter, 1976) that when the enzyme is treated with iodoacetate or some other reagents in the presence of the 'transition-state analogue' complex (i.e. Mg2++ADP+creatine+ nitrate), the thiol groups exhibit differential reactivity. Analysis of these reactions has suggested that differences in reactivity of up to about 7-fold between the groups can be exhibited under these conditions. Astudy of the reaction of the enzyme with various derivatives of iodoacetamide and at different temperatures has revealed further conditions under which differential reactivity of the groups can be exhibited. The reagents used in the study were iodoacetamide, 4iodoacetamidosalicyclic acid, N-(iodoacetylaminoethyl)-5-aminonaphthalene-l-sulphonic acid and 2-[4'-(2"-iodoacetamido)phenyl]aminonaphthalene-6-sulphonic acid. Modification of the enzyme was studied by monitoring the enzyme activity of portions of the reaction mixtures withdrawn at known times and diluted into 0.1 M-glycine/NaOH buffer, pH9.0, containing 2m-dithiothreitol. Control experiments showed in each case that this dilution procedure effectively stopped the reaction. It was also shown, for the reaction of the enzyme with the iodoacetamide derivatives, that the decrease in enzyme activity was proportional to the amount of reagent incorporated, and to the loss of thiol groups whichreactedrapidlywith 5,5'dithiobis-(2-nitrobenzoic acid), and hence with iodoacetamide (Watts, 1973; Price & Hunter, 1976). All reactions were performed in 5Om-Tricine (N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]glycine}/NaOH buffer, pH 8.0, at either 25 or O"C, as indicated. The reactions were analysed as described previously (Price & Hunter, 1976) with the results summarized in Table 1. The results show that the reactions of iodoacetamide with the enzyme at 25°C and at 0°C follow second-order kinetics for at least 85 % of the total reaction, and thus that the two thiol groups react at the same rate as each other. In the case of 4-iodoacetamidosalicylic acid and N-(iodoacetylaminoethyl)-5-aminonaphthalene-l-sulphonic acid,