Diverse interactions between the individual mutations in a double mutant at the active site of staphylococcal nuclease.
In principle, the quantitative effect of a second mutation on a mutant enzyme may be antagonistic, absent, partially additive, additive, or synergistic with respect to the first mutation. Depending on the kinetic or thermodynamic parameter measured, the D21E and R87G mutations of staphylococcal nuclease exhibit four of these five categories of interaction in the double mutant. While Vmax of the R87G single mutant of staphylococcal nuclease is 10(4.8)-fold lower than that of the wild-type enzyme and the Vmax of the D21E single mutant is 10(3.0)-fold below that of wild type, the double mutant D21E + R87G was found to lose a factor of only 10(4.1) in Vmax relative to wild type, rather than the product of the two single mutations (10(7.8)). These results suggest antagonistic structural effects of the individual R87G and D21E mutations. An alternative explanation for the nonadditivity of effects, namely, the separate functioning of these residues in a stepwise mechanism involving the prior attack of water on phosphorus followed by protonation of the leaving group by Arg-87, is unlikely since no enzyme-bound phosphorane intermediate (less than 1% of [enzyme]) was found under steady-state conditions on the R87G mutant by 31P NMR at 242.9 MHz. Like the effects on Vmax, quantitatively similar antagonistic effects of the two mutations were detected on the binding of divalent cations in binary enzyme-Ca2+ and enzyme-Mn2+ complexes and in the ternary enzyme-Ca2(+)-5'-pdTdA complex, suggesting that the effects on Vmax result from antagonistic structural changes at the Ca2+ binding site. Simple additive weakening effects of the two mutations were found on the binding of the substrate 5'-pdTdA, in both the absence and the presence of the divalent cations, Mn2+ and Ca2+. However, synergistic effects of the two mutations were found on the binding of the substrate analogue 3',5'-pdTp, profoundly weakening its binding to the double mutant in both the absence and the presence of divalent cations. Such synergistic effects of the two mutations may result from negative cooperativity or strain in the binding of 3',5'-pdTp to the wild-type enzyme. It is concluded that the quantitative interactions of two active-site mutations of an enzyme can vary greatly depending on which parameter of the enzyme is measured. When the two mutations interact in the same way on several parameters, a common underlying mechanism is suggested.
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
D J
DJ
Weber
E H
EH
Serpersu
D
D
Shortle
A S
AS
Mildvan
D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D008836:Micrococcal Nuclease / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013816:Thermodynamics
D011993:Recombinant Fusion Proteins; D008345:Manganese; D012313:RNA; D004247:DNA; D008836:Micrococcal Nuclease; D002118:Calcium