The effects of amino acid replacements of glycine 20 on conformational stability and catalysis of staphylococcal nuclease

Abstract

Staphylococcal nuclease (SNase) is a well-established model for protein folding studies. Its three-dimensional structure has been determined. The enzyme, Ca 2+, and DNA or RNA substrate form a ternary complex. Glycine 20 is the second position of the first β-turn of SNase, which may serve as the folding initiation site for the SNase polypeptide. To study the role of Gly20 in the conformational stability and catalysis of SNase, three mutants, in which Gly20 was replaced by alanine, valine, or isoleucine, were constructed and studied by using circular dichroism spectra, intrinsic and ANS-binding fluorescence spectra, stability and activity assays. The mutations have little effect on the conformational integrity of the mutants. However, the catalytic activity is reduced drastically by the mutations, and the stability of the protein is progressively decreased in the order G20A<G20V<G20I. Kinetic analysis indicates that the mutant enzymes G20A and G20V show almost 20-fold higher K m Ca values than the wild-type enzyme, and the value for G20I is more than 50-fold higher. K A Ca values indicate more than 17.5-fold weaker binding of Ca 2+ to the G20A and G20V mutants, and more than 39-fold weaker to the G20I mutant, compared to wild-type SNase. The above results suggest that the substitutions at Gly20 cause significantly weaker binding of Ca 2+ in both the binary enzyme-Ca 2+ complex and the ternary complex. However, there is little difference in the values of K m DNA and K S DNA between the mutants and the wild-type enzyme, suggesting that the substitutions at Gly20 have little effect on the binding of DNA substrates to the enzyme. Consistent with the changes in K m Ca and K A Ca , the mutant enzymes G20A, G20V and G20I show about 10 3-, 10 4- and 10 5-fold lower K Cat values than the wild-type enzyme, respectively. These results suggest that Gly20 plays an important role in maintaining a suitable conformation at the active site of the enzyme.