Probing the mechanism of staphylococcal nuclease with unnatural amino acids: kinetic and structural studies

Science. 1993 Sep 17;261(5128):1578-81. doi: 10.1126/science.8103944.

Abstract

Staphylococcal nuclease is an enzyme with enormous catalytic power, accelerating phosphodiester bond hydrolysis by a factor of 10(16) over the spontaneous rate. The mechanistic basis for this rate acceleration was investigated by substitution of the active site residues Glu43, Arg35, and Arg87 with unnatural amino acid analogs. Two Glu43 mutants, one containing the nitro analog of glutamate and the other containing homoglutamate, retained high catalytic activity at pH 9.9, but were less active than the wild-type enzyme at lower pH values. The x-ray crystal structure of the homoglutamate mutant revealed that the carboxylate side chain of this residue occupies a position and orientation similar to that of Glu43 in the wild-type enzyme. The increase in steric bulk is accommodated by a backbone shift and altered torsion angles. The nitro and the homoglutamate mutants display similar pH versus rate profiles, which differ from that of the wild-type enzyme. Taken together, these studies suggest that Glu43 may not act as a general base, as previously thought, but may play a more complex structural role during catalysis.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 2-Aminoadipic Acid / chemistry
  • Amino Acids / chemistry
  • Aminobutyrates / chemistry
  • Arginine / chemistry*
  • Binding Sites
  • Catalysis
  • Glutamates / chemistry*
  • Glutamic Acid
  • Homocysteine / analogs & derivatives
  • Homocysteine / chemistry
  • Hydrogen Bonding
  • Hydrogen-Ion Concentration
  • Kinetics
  • Micrococcal Nuclease / chemistry
  • Micrococcal Nuclease / genetics
  • Micrococcal Nuclease / metabolism*
  • Mutation
  • Plasmids
  • X-Ray Diffraction

Substances

  • Amino Acids
  • Aminobutyrates
  • Glutamates
  • Homocysteine
  • 4-nitro-2-aminobutyric acid
  • 2-Aminoadipic Acid
  • Glutamic Acid
  • 2-amino-5-hydroxyvaleric acid
  • 5-(2-aminoethyl)homocysteine
  • Arginine
  • Micrococcal Nuclease