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Multinuclear magnetic resonance and kinetic studies of single amino acid replacements in staphylococcal nuclease

Conference · · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
OSTI ID:6992411
; ; ; ;

Staphylococcal nuclease hydrolyzes the 5'-phosphate bond of deoxythymidine 5'-p-nitrophenylphosphate to yield p-nitrophenyl phosphate (PNPP) and deoxythymidine in the presence of Ca/sup 2 +/. The PNPP produced can be hydrolyzed to p-nitrophenol and inorganic phosphate by alkaline phosphatase in a coupled assay to provide a chromophore suitable for kinetic studies. By using this assay, the following single amino acid replacements have been characterized at 20 mM Ca/sup 2 +/ and pH 9.25 with the following results (kinetic parameters are expressed relative to those for SNase). SNase, Km = 2.5 mM, V = 1, V/K = 1; substitution of Tyr for Phe at position 85 (Y85F), Km = 61 mM, V/K = 1; H124R, Km = 3.8 mM, V = 3, V/K = 6; H46Y, Km = 2.5 mM, V = 0.78, V/K = 0.66; F76V, Km = 2.4 mM, V = 2.1, and V/K = 2. Only a small perturbation in the kinetic constants is seen for H124R, H46Y, and F76V. Removal of the hydroxyl from tyrosine 85 diminishes the affinity for substrate. Interactions of the wild-type and variant nucleases with metal ions and inhibitors are being investigated by /sup 1/H, /sup 13/C, and /sup 113/Cd NMR.

Research Organization:
Univ. of Wisconsin, Madison
OSTI ID:
6992411
Report Number(s):
CONF-8606151-
Journal Information:
Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 45:6; ISSN FEPRA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
550600 -- Medicine
59 BASIC BIOLOGICAL SCIENCES
62 RADIOLOGY AND NUCLEAR MEDICINE
AROMATICS
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CADMIUM 113
CADMIUM ISOTOPES
CARBON 13
CARBON ISOTOPES
ENZYMES
ESTERASES
EVEN-ODD NUCLEI
HYDROLASES
HYDROXY COMPOUNDS
INTERMEDIATE MASS NUCLEI
ISOMERIC TRANSITION ISOTOPES
ISOTOPES
KINETICS
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
NITRO COMPOUNDS
NITROPHENOL
NMR SPECTRA
NUCLEAR MAGNETIC RESONANCE
NUCLEASES
NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXYGEN COMPOUNDS
PHENOLS
PHOSPHATES
PHOSPHODIESTERASES
PHOSPHORUS COMPOUNDS
RADIOISOTOPES
REACTION KINETICS
RESONANCE
SPECTRA
STABLE ISOTOPES
STAPHYLOCOCCUS
YEARS LIVING RADIOISOTOPES