Site-directed mutants of staphylococcal nuclease. Detection and localization by 1H 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. We have used primer-directed, site-specific mutagenesis to generate aspartate, glutamine, asparagine, alanine, and serine substitutions for this residue. The Vmax/Km 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 1H NMR spectral properties of the mutants suggest that such interpretations are ambiguous. All five mutants have higher melting temperatures for thermal denaturation than the wild-type enzyme, suggesting that the mutants have enhanced thermal stabilities relative to the wild-type enzyme. Chemical shift changes relative to the wild type are observed in both the aromatic and upfield-shifted methyl group regions of the 1H 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 comparisons 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.(ABSTRACT TRUNCATED AT 250 WORDS)
D001483:Base Sequence; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008836:Micrococcal Nuclease / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D011489:Protein Denaturation; D013210:Staphylococcus / Q000201:enzymology / Q000235:genetics; D013698:Templates, Genetic
D005971:Glutamates; D018698:Glutamic Acid; D008836:Micrococcal Nuclease