Use of methanethiolation to investigate the catalytic role of sulphydryl groups in rabbit skeletal muscle pyruvate kinase.
Incubation of rabbit skeletal muscle pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) with methyl methanethiosulphonate resulted in the time- and inhibitor concentration-dependent loss of enzyme activity. Substrates or products of the catalytic reaction prevented the loss of activity caused by methanethiolation. Their effectiveness as protecting agents was placed in the order ADP greater than ATP greater than Mg2+ greater than phosphoenolpyruvate greater than pyruvate. The essential catalytic cation, K+, had no effect on the methanethiolation reaction. [Me-3H]Methanethiosulphonate modified all the available cysteine thiol groups which correlated to the incorporation of four SC3H3 groups per protomer. Four radioactive peptides were obtained on tryptic peptide mapping. When methanethiolation was carried out in the presence of Mg2+ alone or with Mg2+ and ATP together, then only three SC3H3 groups were incorporated into each subunit. If MgATP protected methanethiolated pyruvate kinase was reacted with iodo[2-3H]acetic acid then 1.37 +/- 0.2 groups per protomer were carboxymethylated. 70% of the radioactivity was located in a single peptide on tryptic peptide mapping. This peptide was isolated and contained the segment carboxymethyl cysteine (Glx, Asx, Ser) Arg. Collectively these data indicate that although all thiol groups are equally accessible to methyl methanethiosulphonate, only a single thiol group participates in the catalytic event. An additional role in the maintenance of structure for this thiol group was also shown in studied of reduction and thermal denaturation of the enzyme.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D004229:Dithiothreitol / Q000494:pharmacology; D007461:Iodoacetates / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008698:Mesylates / Q000494:pharmacology*; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology*; D009132:Muscles / Q000201:enzymology*; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D011489:Protein Denaturation; D011770:Pyruvate Kinase / Q000378:metabolism*; D011773:Pyruvates / Q000494:pharmacology; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013886:Thiosulfonic Acids / Q000494:pharmacology
D007461:Iodoacetates; D008698:Mesylates; D011773:Pyruvates; D013438:Sulfhydryl Compounds; D013886:Thiosulfonic Acids; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D000255:Adenosine Triphosphate; D008741:Methyl Methanesulfonate; D011770:Pyruvate Kinase; D008274:Magnesium; D004229:Dithiothreitol