J. Biochem, 1978, Vol. 83, No. 3 789-793
© 1978 Japanese Biochemical Society
research-article |
pH-Profiles of the Kinetic Parameters of a Minor Ribonuclease from Aspergillus saitoi
Department of Microbiology, Hoshi College of Pharmacy Ebara, Shinagawa-ku, Tokyo 142
In order to investigate the nature of amino acid residues involved in the active site of a ribonuclease from Aspergillus saitoi, the pH-profiles of kinetic parameters of RNase Ms were measured. (1) The pH dependences of kinetic parameters were measured using 2', 3'-CCMP and 2‘, 3’-cUMP as substrates. Analyses of the results indicated the presence of three functional groups having pKa values of ca. 2.7 4.5, and 6.75 in the active site of RNase Ms. (2) The pH dependence of the inhibition constant of 2', (3')-AMP gave a pattern similar to the pK1-pH profiles of the substrate described above, again indicating the involvement of three functional groups in the active site. (3) The inhibition constant of adenosine changed only slightly with change in pH, and the functional group having pKa about 6.75 was not observed. The results indicate the interaction of this group with the phosphate moiety of the nucleotide. (4) A similar dependence of kinetic parameters of RNase Ms Using three substrates of the cyclization step was obtained. (5) By analyzing the pH-profiles of pKm and pK1, it was concluded that the active site of RNase Ms contains at least three functional groups, a histidine pKa about 6.75), a histidine or carboxyl group (pKa 4.0–4.5) and a carboxyl group (pKa about 2.7).