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Contribution of active site residues to the activity and thermal stability of ribonuclease Sa

¹ Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
² Department of Biology, Grove City College, Grove City, Pennsylvania 16127, USA
³ Department of Medical Biochemistry and Genetics, Texas A&M University, College Station, Texas 77843, USA

Reprint requests to: C. Nick Pace, Department of Medical Biochemistry and Genetics, Texas A&M University, College Station, TX 77843, USA; e-mail: nickpace{at}tamu.edu; fax: (979) 847-9481.

We have used site-specific mutagenesis to study the contribution of Glu 74 and the active site residues Gln 38, Glu 41, Glu 54, Arg 65, and His 85 to the catalytic activity and thermal stability of ribonuclease Sa. The activity of Gln38Ala is lowered by one order of magnitude, which confirms the involvement of this residue in substrate binding. In contrast, Glu41Lys had no effect on the ribonuclease Sa activity. This is surprising, because the hydrogen bond between the guanosine N1 atom and the side chain of Glu 41 is thought to be important for the guanine specificity in related ribonucleases. The activities of Glu54Gln and Arg65Ala are both lowered about 1000-fold, and His85Gln is totally inactive, confirming the importance of these residues to the catalytic function of ribonuclease Sa. In Glu74Lys, k_cat is reduced sixfold despite the fact that Glu 74 is over 15 Å from the active site. The pH dependence of k_cat/K_M is very similar for Glu74Lys and wild-type RNase Sa, suggesting that this is not due to a change in the pK values of the groups involved in catalysis. Compared to wild-type RNase Sa, the stabilities of Gln38Ala and Glu74Lys are increased, the stabilities of Glu41Lys, Glu54Gln, and Arg65Ala are decreased and the stability of His85Gln is unchanged. Thus, the active site residues in the ribonuclease Sa make different contributions to the stability.

Keywords: Ribonuclease Sa; active-site mutants; catalytic activity; specificity; thermal stability

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