Role of Phe120 in the activity and structure of bovine pancreatic ribonuclease A.
Phenylalanine120 is a candidate residue juxtaposing catalytic His12 and His119 in ribonuclease A (RNase A). To clarify its role in construction of the catalytic center, Phe120 was replaced by alanine, tryptophan, leucine, or glutamic acid by site-directed mutagenesis. The transphosphorylation and hydrolysis activities of the mutant RNase As, respectively, toward cytidinyl 3',5' adenosine (CpA) and cytidine 2',3' cyclic monophosphate (C>p) were compared with those of the wild type enzyme. The Km values of the two reactions increased markedly with slight changes in the Kcat values. The pKe values of His12 and His119 in the wild type and mutant enzymes, estimated from the pH dependence of the kcat/Km values, showed little change. The rate of carboxymethylation was reduced markedly by the mutations. The Ki values of the phosphate anion as to hydrolysis activity increased only slightly when Phe120 was replaced by leucine, tryptophan, or alanine. These findings suggest that Phe120 participates in the binding of the substrate, juxtaposing His12 and His119, and in stabilizing the transition state intermediate in the hydrolysis reaction. Furthermore, the decreases in the thermal denaturation temperatures of all the mutants, particularly F120E, indicate that Phe120 also helps maintain the conformational stability of RNase A.
Laboratory of Biomacromolecular Chemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.
N
N
Tanimizu
H
H
Ueno
R
R
Hayashi
D000818:Animals; D001667:Binding, Competitive; D002384:Catalysis; D002417:Cattle; D004926:Escherichia coli; D006639:Histidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007461:Iodoacetates / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010179:Pancreas / Q000201:enzymology*; D010649:Phenylalanine / Q000737:chemistry / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism*
D007461:Iodoacetates; D010649:Phenylalanine; D006639:Histidine; D012259:Ribonuclease, Pancreatic