Carboxymethylation of an active site glutamic acid residue of ribonuclease F1 iodoacetate.
Ribonuclease (RNase) F1 was inactivated by incubation with an excess amount of iodoacetate at pH 5.5, 37 degrees C according to pseudo first-order kinetics. It was protected to various degrees, from inactivation by nucleotides, among which guanosine 2'-phosphate was most effective. The pseudo first-order rate constant was proportional to the reagent concentration, indicating that the reaction in reality follows second-order kinetics. The second-order rate constant was determined to be 25 x 10(-4) M-1 s-1. The inactivation rate was maximal at pH 5.5-6.0. When iodo[2-14C]acetate was used as the reagent, the stoichiometry of incorporation was determined to be 1.1 mol carboxymethyl group per mol of RNase F1 and glutamic acid residue 58 was assigned as the site of modification.
Yoshida
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Department of Chemistry, Faculty of Science, Tohoku University, Sendai, Japan.
Hanazawa
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H
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D004722:Endoribonucleases; D004789:Enzyme Activation / Q000187:drug effects; D005670:Fusarium / Q000201:enzymology*; D005971:Glutamates; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D007461:Iodoacetates; D019807:Iodoacetic Acid; D007700:Kinetics; D008745:Methylation; D008956:Models, Chemical; D009711:Nucleotides; D012697:Serine Endopeptidases
D000596:Amino Acids; D005971:Glutamates; D007461:Iodoacetates; D009711:Nucleotides; D018698:Glutamic Acid; D004722:Endoribonucleases; C021885:ribonuclease F; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D019807:Iodoacetic Acid