Conformational stability, flexibility, and steady-state activity of Ribonuclease T1 variants

Abstract

The steady-state kinetic parameters for GpC hydrolysis by wild-type RNase T1 and 14 single-residue mutants were determined at pH 7.0 and 25 'C. These mutants comprise a 4 kcal/mol range in global stability under similar conditions (Shirley et al., 1992; Thomson et al., 1989). We observe a relationship between the variants' conformational stability and the determined Kcat/Km values. With the exception of three mutations made to the active site, differences from wild-type activity are believed to be a result of changes in conformational dynamics. The mutants can be classified into three categories: those with increased stability and decreased activity from wild-type values; those with decreased stability and decreased activity; and those with decreased stability and increased activity. The fourth possibility, increased stability and activity was not observed, possibly due to decreases in flexibility required for binding and catalysis. Variation in activity among mutants of similar stability demonstrates the context dependence of mutational effects. Although results are preliminary, this model system may reflect a universal relationship between stability and activity.