A CATALYTIC FUNCTION FOR THE STRUCTURALLY CONSERVED RESIDUE PHE-100 OF RIBONUCLEASE T-1

The function of the conserved Phe 100 residue of RNase T-1 (EC 3.1.27. 3) has been investigated by site-directed mutagenesis and X-ray crysta llography. Replacement of Phe 100 by alanine results in a mutant enzym e with k(cat) reduced 75-fold and a small increase in K-m for the dinu cleoside phosphate substrate GpC. The Phe 100 Ala substitution has sim ilar effects on the turnover rates of GpC and its minimal analogue GpO Me, in which the leaving cytidine is replaced by methanol. The contrib ution to catalysis is independent of the nature of the leaving group, indicating that Phe 100 belongs to the primary site. The contribution of Phe 100 to catalysis may result from a direct van der Waals contact between its aromatic ring and the phosphate moiety of the substrate. Phe 100 may also contribute to the positioning of the pentacovalent ph osphorus of the transition state, relative to other catalytic residues . If compared to the corresponding wild-type data, the structural impl ications of the mutation in the present crystal structure of Phe 100 A la RNase T-1 complexed with the specific inhibitor 2'-GMP are restrict ed to the active site. Repositioning of 2'-GMP, caused by the Phe 100 Ala mutation, generates new or improved contacts of the phosphate moie ty with Arg 77 and His 92. In contrast, interactions with the Glu 58 c arboxylate appear to be weakened. The effects of the His 92 Gln and Ph e 100 Ala mutations on GpC turnover are additive in the corresponding double mutant, indicating that the contribution of Phe 100 to catalysi s is independent of the catalytic acid His 92. The present results lea d to the conclusion that apolar residues may contribute considerably t o catalyze conversions of charged molecules to charged products, invol ving even more polar transition states.