Kinetic and conformational effects of lysine substitutions for arginines 35 and 87 in the active site of staphylococcal nuclease.
The high-resolution X-ray crystal structure of staphylococcal nuclease (SNase) suggests that the guanidinium groups of Arg 35 and Arg 87 participate as electrophilic catalysts in the attack of water on the substrate phosphodiester. Both arginine residues have been replaced with "conservative" lysine residues so that both the importance of these residues in catalysis and the effect of changes in electrostatic interactions on active site conformation can be assessed. The catalytic efficiencies of R35K and R87K are decreased by factors of 10(4) and 10(5) relative to wild-type SNase, with R87K showing a very significant reduction in its affinity for both DNA substrate and the competitive inhibitor thymidine 3',5'-bisphosphate (pdTp). The thermal denaturation behavior of both mutant enzymes differs from that of wild type both in the absence and in the presence of the active site ligands Ca2+ and pdTp. Both the 1H NMR chemical shifts and interresidue nuclear Overhauser effects (NOEs) of residues previously assigned to be in the hydrophobic core of SNase are altered in R35K and R87K. These observations, similar to those recently reported by our laboratories for substitutions for Glu 43 [Hibler, D. W., Stolowich, N. J., Reynolds, M. A., Gerlt, J. A., Wilde, J. A., & Bolton, P. H. (1987) Biochemistry 26, 6278; Wilde, J. A., Bolton, P. H., Dell'Acqua, M., Hibler, D. W., Pourmotabbed, T., & Gerlt, J. A. (1988) Biochemistry 27, 4127], suggest that lysine substitutions are not conservative in SNase and disrupt the conformation of the active site.(ABSTRACT TRUNCATED AT 250 WORDS)
Pourmotabbed
T
T
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Dell'Acqua
M
M
Gerlt
J A
JA
Stanczyk
S M
SM
Bolton
P H
PH
D001120:Arginine / Q000235:genetics*; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D008239:Lysine / Q000235:genetics*; D009682:Magnetic Resonance Spectroscopy; D008836:Micrococcal Nuclease / Q000235:genetics*; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation
D001120:Arginine; D008836:Micrococcal Nuclease; D008239:Lysine; D002118:Calcium