Semisynthetic fluorohydrolases prepared by chemical modification of ribonuclease.
A process for conformational modification of protein, which we have previously reported, was investigated as a means of generating fluorohydrolase activity in bovine ribonuclease (RNase). The resulting modified RNase had catalytic activity that depended upon the chosen modifier. Bovine pancreatic ribonuclease, modified by addition of hexamethylphosphoramide (HMPA) at pH 3, was derivatized with diimidates of chain lengths from C1 to C8. The derivative with the highest activity was obtained when RNase was crosslinked with dimethyl pimelimidate (C5). This derivative, which was active over a pH range of 6.5 to 8.0 with an optimum pH of 7.4, hydrolyzed phenylmethylsulfonylfluoride (PMSF) and the potent acetylcholinesterase inhibitor, diisopropyl phosphorofluoridate (DFP). The mean fluorohydrolase activity for four preparations using dimethyl pimelimidate was 0.8 +/- 0.2 U mg-1. Gel filtration on G-75 Sephadex and SDS-polyacrylamide gel electrophoresis showed components having a molecular weight of 13,000 and 27,000, with activity restricted to the 27,000 molecular weight fraction. After gel filtration, the specific activity was 9.1 +/- 2.4 U mg-1, resulting in a molecular activity of 125 min-1. The mechanism of this unique transformation of RNase into a fluorohydrolase is not known, nor has the location of the active site been determined.
Albert
D E
DE
Anatrace, Inc., Maumee, OH 43537.
Douglas
M B
MB
Hintz
M A
MA
Youngen
C S
CS
Keyes
M H
MH
D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents; D005949:Glucose Oxidase / Q000138:chemical synthesis / Q000378:metabolism; D006593:Hexokinase / Q000138:chemical synthesis / Q000378:metabolism; D006867:Hydrolases / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D007096:Imidoesters; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology; D011487:Protein Conformation; D012260:Ribonucleases / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*
D003432:Cross-Linking Reagents; D007096:Imidoesters; C029326:dimethyl pimelimidate; D005949:Glucose Oxidase; D006593:Hexokinase; D006867:Hydrolases; D012260:Ribonucleases