Inactivation of pig muscle 3-phosphoglycerate kinase by thiol modification depends on reagent size.
Two thiol groups (out of the total seven) of pig muscle 3-phosphoglycerate kinase can be alkylated in 0.1 M Tris/Hcl buffer, pH 7.5, at 20 degrees C either with methyl iodide or with iodoacetamide in a second-order reaction with rate constants 0.05 +/- 0.02 M-1S-1 and 0.23 +/- 0.05 M-1S-1 respectively. The slow reaction of the remaining five thiols with Ellman's reagent (Nbs2), which requires the unfolding of the protein, is not affected by the nature of the alkylating reagent. While methylation of the two reactive thiols does not affect either the specific activity of the enzyme or the Km values of the substrates, carboxamidomethylation is accompanied by the loss of enzymic activity. Both the methylated and the carboxamidomethylated enzymes bind 3-phosphoglycerate or MgATP practically with the same binding constants as the native enzyme, as detected by fluorimetric titration of enzymes complexed with 1-anilinonaphthalenesulfonate (ANS). Thus, inactivation during carboxamidomethylation cannot be due to changes in the substrate-binding ability of the enzyme. The substrate-caused changes in the fluorescence emission spectrum of ANS bound to the carboxamidomethylated enzyme are different from the changes observed with the native enzyme. The fluorescence properties of the methylated enzyme do not differ from those of the native enzyme. These differences may reflect the different mode of substrate binding to the carboxamidomethylated enzyme as compared to the native or the methylated one. Thus, the special steric requirements of the enzymic reaction are possibly not fulfilled after carboxamidomethylation. The presence of the equilibrium mixture of substrates lessens the reactivity of the fast-reacting thiols towards both alkylating agents, a finding that indicates the effects of substrates on the local conformation around these groups.
D000477:Alkylating Agents / Q000494:pharmacology*; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D004228:Dithionitrobenzoic Acid; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010735:Phosphoglycerate Kinase / Q000037:antagonists & inhibitors*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D013552:Swine
D000477:Alkylating Agents; D007202:Indicators and Reagents; D013439:Sulfhydryl Reagents; D004228:Dithionitrobenzoic Acid; D010735:Phosphoglycerate Kinase