PROBING THE MECHANISM OF STAPHYLOCOCCAL NUCLEASE WITH UNNATURAL AMINO-ACIDS - KINETIC AND STRUCTURAL STUDIES

Staphylococcal nuclease is an enzyme with enormous catalytic power, ac celerating phosphodiester bond hydrolysis by a factor of 10(16) over t he spontaneous rate. The mechanistic basis for this rate acceleration was investigated by substitution of the active site residues Glu43, Ar g35, and Arg87 With unnatural amino acid analogs. Two Glu43 mutants, o ne containing the nitro analog of glutamate and the other containing h omoglutamate, retained high catalytic activity at pH 9.9, but were les s active than the wild-type enzyme at lower pH values. The x-ray cryst al structure of the homoglutamate mutant revealed that the carboxylate side chain of this residue occupies a position and orientation simila r to that of Glu43 in the wild-type enzyme. The increase in steric bul k is accommodated by a backbone shift and altered torsion angles. The nitro and the homoglutamate mutants display similar pH versus rate pro files, which differ from that of the wild-type enzyme. Taken together, these studies suggest that Glu43 may not act as a general base, as pr eviously thought, but may play a more complex structural role during c atalysis.