| Abstract: | 4-Arsono-2-nitrofluorobenzene reacts selectively at the anion binding site of bovine pancreatic ribonuclease A. The major derivative is the inactive 41-(4-arsono-2-nitrophenyl) ribonuclease A (45% yield). Additional products are 1-alpha-(4-arsono-2-nitrophenyl) ribonuclease A (11% yield) which is enzymatically active and the disubstituted, inactive 1,41-bis-(4-arsono-2-nitrophenyl) ribonuclease A (25% yield). 2' (3')-O-Bromoacetyluridine reacts with 41-(4-arsono-2-nitrophenyl) ribonuclease A exclusively at the histidine-12 residue at a rate which is approximately one-fourth the rate observed with the unmodified enzyme. Saturation kinetics are observed and the dissociation constant for the protein-inhibitor complex is 0.096 +/- 0.023 M. The first-order unimolecular decomposition constant for complex breakdown is 8.9 +/- 2.9 X 10(-4) s-1. 2'-Bromoacetamido-2'-deoxyuridine reacts with 41-(4-arsono-2-nitrophenyl) ribonuclease A 25 times more slowly than 2'(3')-O-bromoacetyluridine. Bromoacetate reacts with 41-(4-arsono-2-nitrophenyl) ribonuclease A predominantly at the histidine-119 residue at a rate 45 times less than that found for the unmodified enzyme. The results of the alkylation studies imply that the dianionic arsonate does not occupy the phosphate binding site in the enzyme but is sufficiently proximate to account for a decrease in bromoacetate binding as well as a reduction in the nucleophilic reactivity of histidine-12 and -119. All these effects may be accounted for in terms of a local electrostatic perturbation of the active site region by the arsononitrophenyl group. |
