Site-directed mutants of staphylococcal nuclease. Detection and localization by /sup 1/H NMR spectroscopy of conformational changes accompanying substitutions for glutamic acid-43
The high-resolution X-ray crystal structure of staphylococcal nuclease suggests that the ..gamma..-carboxylate group of Glu-43 is directly involved in catalysis as a general base that facilitates the attack of water on the substrate phosphodiester. The authors have used primer-directed, site-specific mutagenesis to generate aspartate, glutamine, asparagine, alanine, and serine substitutions for this residue. The V/sub max//K/sub m/ for the aspartate mutant is reduced 1400-fold and the values for the charge-neutral mutations are reduced 5000-fold relative to the wild-type enzyme. Although these reductions in catalytic efficiency might appear useful in quantitatively estimating the importance of general basic catalysis in the reaction catalyzed by the wild-type enzyme, the thermal stabilities and /sup 1/H NMR spectral properties of the mutants suggest that such interpretations are ambiguous. Chemical shift changes relative to the wild type are observed in both the aromatic and upfield-shifted methyl group regions of the /sup 1/H NMR spectra of the aspartate and serine mutants, suggesting the presence of conformational differences between the wild-type and mutant enzymes. That these conformational differences may be large enough to be mechanistically relevant is suggested by comparison of the magnitudes of nuclear Overhauser effect (NOE) correlations between the aromatic and upfield-shifted methyl group regions observed via two-dimensional nuclear Overhauser effect correlation spectroscopy. The aromatic protons involved in NOE correlations that differ in intensity in the wild-type and mutant proteins have been localized to the three phenylalanine residues present in the protein. Since these phenylalanine residues are at least 15 A removed from the position of Glu-43 in the structure of the wild-type enzyme, the substitutions for Glu-43 are accompanied by global changes in the conformation of the protein molecule.
- Research Organization:
- Univ. of Maryland, College Park
- OSTI ID:
- 5400306
- Journal Information:
- Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:19; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
Similar Records
Kinetic and conformational effects of lysine substitutions for arginines 35 and 87 in the active site of Staphylococcal nuclease
Proton nuclear magnetic resonance studies on the wild-type and single amino acid substituted tryptophan synthase. cap alpha. -subunits
Related Subjects
62 RADIOLOGY AND NUCLEAR MEDICINE
AMINO ACIDS
BACTERIA
BARYONS
CARBOXYLIC ACIDS
CONFORMATIONAL CHANGES
DEUTERIUM COMPOUNDS
ELEMENTARY PARTICLES
ENZYMES
ESTERASES
FERMIONS
GLUTAMIC ACID
HADRONS
HYDROGEN COMPOUNDS
HYDROLASES
MAGNETIC RESONANCE
MICROORGANISMS
MUTANTS
NMR SPECTRA
NUCLEAR MAGNETIC RESONANCE
NUCLEASES
NUCLEONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
PHOSPHODIESTERASES
PROTONS
RESONANCE
SPECTRA
STAPHYLOCOCCUS