The Effect of Elimination of Amino Acids from the Carboxyl-terminal of the Minor Ribonuclease from Aspergillus saitoi by Carboxypeptidase A on the Enzymatic Activity

Abstract

In order to investigate whether carboxyl-terminal amino acid is involved in the active site of a base non-specific ribonuclease from Asp. saitoi (RNase Ms), removal of carboxyl-terminal amino acid residues by digestion with carboxypeptidase A was investigated. By 24 hours digestion, about 3 serine residues and 1.0 alanine residue were removed from RNase Ms and its activity decreased to about 70% of the native enzyme so far as measured with ribonucleic acid (RNA) as a substrate. The pH optimum and K_m of carboxypeptidase treated RNase Ms (CP-RNase Ms) were very similar to those of native RNase Ms so far as measured with RNA as a substrate. However, K_m of CP-RNase Ms using ApC as a substrate seemed to be larger than that of native RNase Ms. The large increase in K_m value was not observed in the early stage of digestion where about 3 serine residues were removed. The gross structure of CP-RNase Ms was quite similar to that of the native RNase Ms by judging from circular dichroism spectrum at wavelength between 230-205 nm. From the results described above, it was concluded that carboxyl-terminal amino acid was not involved directly in the active site of RNase Ms.