The Structure and Function of Ribonuclease T1

Publisher Website
Google Scholar
Abstract
1. The interaction of ribonuclease T1 [EC 2. 7. 7. 26] with its substrate analogs has been investigated by a gel filtration method. The enzyme bound maximally one molecule of 3′-GMP per molecule of protein, indicating the presence of one specific binding site in the enzyme molecule. 2. At pH 5.5 and 25°C, the apparent binding strengths of the substrate analogs were in the order: 2′-GMP>3′-GMP>3′, 5′-GDP>9-(2′-hydroxyethyl)guanine 2′-phosphate> 5′-GMP>guanosine>8-bromoguanosine>3′-AMP> deoxyguanosine> 5′-1-MeGMP> 3′-CMP, 3′-UMP, guanine and the other eight nucleosides examined. The results show the importance of the integrity of the guanine, ribose, and phosphate portions for the binding. The importance of the N-1 and N-7 positions and the 2-amino and 6-oxo (or hydroxy) groups in the guanine portion for the specific binding was also suggested. 3. The optimum pH for the binding of 3′-GMP and guanosine to the enzyme was around pH 5. The pH dependence of the binding of these analogs to the enzyme indicated the implication in the binding of at least two groups in the enzyme with pKa values of about 3.5 and 7, respectively, presumably a carboxyl and an imidazole groups. The significant loss of the binding ability toward 3′-GMP at temperatures above 40°C or in the presence of urea demonstrates the importance of the native conformation of the enzyme for the binding. The effects of some added substances on the binding were also investigated. 4. The Glu-58-carboxymethylated, inactive enzyme retained a considerable binding ability toward 3′-GMP and moreover it possessed almost the same binding ability toward guanosine over a wide pH range as that of the native enzyme. These results appear to indicate that Glu-58 is part of the catalytic site rather than part of the binding site, although the latter possibility cannot be excluded. The binding ability toward 3′-GMP was lowered significantly by the modification of Arg-77 with phenylglyoxal and also by the cleavage of the disulfide bonds with β-mercaptoethanol. Arginine-77 may be the phosphate binding site or may be situated near the active site.

This publication has 0 references indexed in Scilit: