Modification of a major ribonuclease from Aspergillus saitoi with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide.
In order to investigate the role of carboxyl groups of a base non-specific ribonuclease from Aspergillus saitoi [EC 3.1.27.1] (RNase M, molecular weight 36,000), the modification of RNase M with a water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide(CMC), was studied. The inactivation of RNase M proceeded almost linearly with the incorporation of about 9.5 CMC moieties. The peptide backbone structure of the modified RNase M was practically the same as that of the native RNase M, as assessed from the CD spectra in the region of 200-250 nm. In the presence of competitive inhibitors, adenosine, and cytidine, inactivation of RNase M by CMC was partially inhibited. In the presence of cytidine (1 M), the modification of about 4 carboxyl groups of RNase M proceeded with a slight loss of enzymatic activity (ca. 20%). Further modification inactivated RNase M with the incorporation of ca. 4-5 CMC without any detectable intramolecular peptide bond formation. Therefore, it was concluded that carboxyl groups responsible for enzymatic activity were included among these carboxyl groups protected by cytidine. The logarithm of the half-live of the inactivation of RNase M by CMC was a linear function of log[CMC] with a slope of minus one, indicating that at least one carboxyl group among the modified ones may be essential for catalysis. The digestion of CMC-modified RNase M with carboxypeptidase A eliminated the carboxyl terminal group from the site of CMC modification.
D000577:Amides / Q000032:analysis; D000596:Amino Acids / Q000032:analysis; D001230:Aspergillus / Q000201:enzymology*; D003028:CME-Carbodiimide / Q000031:analogs & derivatives / Q000494:pharmacology*; D002234:Carbodiimides / Q000494:pharmacology*; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D055598:Chemical Phenomena; D002621:Chemistry; D004722:Endoribonucleases / Q000037:antagonists & inhibitors*; D006868:Hydrolysis; D009705:Nucleosides
D000577:Amides; D000596:Amino Acids; D002234:Carbodiimides; D009705:Nucleosides; D003028:CME-Carbodiimide; D004722:Endoribonucleases; C022538:ribonuclease M; D002268:Carboxypeptidases; D043422:Carboxypeptidases A