Kinetic and conformational effects of lysine substitutions for arginines 35 and 87 in the active site of Staphylococcal nuclease
- Univ. of Maryland, College Park (USA)
- Wesleyan Univ., Middletown, CT (USA)
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{sup 4} and 10{sup 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{prime},5{prime}-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 Ca{sup 2+} and pdTp. Both the {sup 1}H 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 suggest that lysine substitutions are not conservative in SNase and disrupt the conformation of the active site. Thus, the kinetic properties of R35K and R87K cannot be used to describe the roles of Arg 35 and Arg 87 in catalysis by the wild-type enzyme. Furthermore, on the basis of the effects of these conservative substitutions for Arg 35 and Arg 87. The authors predict that other substitutions for these residues, including neutral substitutions, are also likely to cause conformational alterations in the active site.
- OSTI ID:
- 6276377
- Journal Information:
- Biochemistry; (USA), Vol. 29:14; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
NUCLEASES
MOLECULAR STRUCTURE
ARGININE
BIOCHEMICAL REACTION KINETICS
CATALYSIS
CHEMICAL SHIFT
CONFORMATIONAL CHANGES
CRYSTALLOGRAPHY
LIGANDS
LYSINE
MUTAGENESIS
NUCLEAR MAGNETIC RESONANCE
PROTEIN DENATURATION
PROTONS
STAPHYLOCOCCUS
SUBSTRATES
X-RAY DIFFRACTION
AMINO ACIDS
BACTERIA
BARYONS
CARBOXYLIC ACIDS
COHERENT SCATTERING
DIFFRACTION
ELEMENTARY PARTICLES
ENZYMES
ESTERASES
FERMIONS
HADRONS
HYDROLASES
KINETICS
MAGNETIC RESONANCE
MICROORGANISMS
NUCLEONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
PHOSPHODIESTERASES
REACTION KINETICS
RESONANCE
SCATTERING
550201* - Biochemistry- Tracer Techniques