The structure and function of ribonuclease T1. XX. Specific inactivation of ribonuclease T1 by reaction with tosylglycolate.
1. Ribonuclease T1 [EC 3.1.4.8] was inactivated by reaction with tosylglycolate (carboxymethyl rho-toluenesulfonate). At pH 5.5 and 8.0, alkylation of the gamma-carboxyl group of glutamic acid-58 appeared to be the predominant reaction and the major cause of inactivation by tosylglycolate, as in the case of the iodoacetate reaction, although the rate of inactivation was slower than that by iodoacetate. At pH 8.0, histidine residues were also alkylated to some extent. 2. The maximal rate of inactivation was observed at around pH 5.5 and the pH dependence of the rate of inactivation suggested the implication of two groups in the reaction, with apparent pKa values of about 3-4 (possibly histidine residue(s)). 3. In the presence of substrate analogs, ribonuclease T1 was markedly protected from inactivation by tosylglycolate at pH 5.5. The extent of protection corresponded to the binding strength of the substrate analog, except for guanosine. Ribonuclease T1 was much less protected from inactivation by guanosine than by 3'-AMP or 3'-CMP, which has a lower binding strength toward ribonuclease T1. This may indicate that glutamic acid-58 is situated in the catalytic site, at which the phosphate moiety of these nucleotides directly interacts. 4. Enzyme which had been extensively inactivated with tosylglycolate at pH 5.5 scarcely reacted with iodoacetate at pH 5.5, suggesting that these reagents react at the same site, i.e. glutamic acid-58. On the other hand, enzyme which had been inactivated almost completely with tosylglycolate at pH 8.0 still reacted with iodoacetate to some extent at pH 8.0, and the modes of reaction of tosylglycolate and iodoacetate toward ribonuclease T1 appeared to be somewhat different.
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D003568:Cytidine Monophosphate / Q000494:pharmacology; D005971:Glutamates; D006016:Glycolates / Q000494:pharmacology*; D006150:Guanine Nucleotides / Q000494:pharmacology; D006151:Guanosine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007461:Iodoacetates / Q000494:pharmacology; D006163:Ribonuclease T1 / Q000037:antagonists & inhibitors*; D012260:Ribonucleases / Q000037:antagonists & inhibitors*; D014105:Tosyl Compounds / Q000494:pharmacology*
D000596:Amino Acids; D005971:Glutamates; D006016:Glycolates; D006150:Guanine Nucleotides; D007461:Iodoacetates; D014105:Tosyl Compounds; D006151:Guanosine; D000249:Adenosine Monophosphate; D012260:Ribonucleases; D006163:Ribonuclease T1; D003568:Cytidine Monophosphate