Contribution of a highly conserved serine residue to ribonuclease activity and stability

ORGN 650

R. Jeremy Johnson, jjohnson@biochem.wisc.edu, Shawn R. Lin, and Ronald T. Raines, raines@biochem.wisc.edu. Departments of Biochemistry and Chemistry, University of Wisconsin - Madison, 433 Babcock Dr, Madison, WI 53706
Ribonuclease A is perhaps the most studied enzyme. Recently, the essential residues for catalysis by RNase A were determined with a genetic selection, which identified multiple residues that had previously not been recognized as essential to catalysis. One such residue is Ser75, which is 100% conserved among 13 RNase A homologs. Its hydroxymethyl side chain is buried and donates a hydrogen bond to the main chain oxygen of Ile106, a folding core residue. By screening a saturation mutagenesis library at position 75 over a range of temperatures, we observed that the hydrogen bond donated is not as important as is the van der Waal's radius of the amino acid. Moreover, substitution at position 75 does not greatly affect catalysis, but rather strongly influences the thermal stability of the ribonuclease. Thus, Ser75 of RNase A provides new insight into the contribution of buried polar residues to the stability and activity of enzymes.