Publication Date:
1993-09-17
Description:
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.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Judice, J K -- Gamble, T R -- Murphy, E C -- de Vos, A M -- Schultz, P G -- GM 14012-02S1/GM/NIGMS NIH HHS/ -- R01 GM49220/GM/NIGMS NIH HHS/ -- T32GM-08388/GM/NIGMS NIH HHS/ -- New York, N.Y. -- Science. 1993 Sep 17;261(5128):1578-81.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Chemistry, University of California, Berkeley 94720.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/8103944" target="_blank"〉PubMed〈/a〉
Keywords:
2-Aminoadipic Acid/chemistry
;
Amino Acids/chemistry
;
Aminobutyrates/chemistry
;
Arginine/*chemistry
;
Binding Sites
;
Catalysis
;
Glutamates/*chemistry
;
Glutamic Acid
;
Homocysteine/analogs & derivatives/chemistry
;
Hydrogen Bonding
;
Hydrogen-Ion Concentration
;
Kinetics
;
Micrococcal Nuclease/chemistry/genetics/*metabolism
;
Mutation
;
Plasmids
;
X-Ray Diffraction
Print ISSN:
0036-8075
Electronic ISSN:
1095-9203
Topics:
Biology
,
Chemistry and Pharmacology
,
Computer Science
,
Medicine
,
Natural Sciences in General
,
Physics
