A SINGLE AMINO-ACID SUBSTITUTION CHANGES RIBONUCLEASE-4 FROM A URIDINE-SPECIFIC TO A CYTIDINE-SPECIFIC ENZYME

The structural features underlying the strong uridine specificity of r ibonuclease 4 (RNase 4) are largely unknown. It has been hypothesized that the negatively charged alpha-carboxylate is close to the pyrimidi ne binding pocket, due to a unique C-terminal deletion. This would sup press the cleavage of cytidine-containing substrates [Zhou, H.-M., and Strydom, D. J. (1993) Eur. J. Biochem. 217, 401-410]. Replacement of the alpha-carboxylate by an alpha-carboxamide in a fragment complement ation system decreased both (k(cat)/K-m)(CpA) and (k(cat)/K-m)(UpA), t hus refuting the hypothesis. However, model building showed that the d eletion allowed the side chain of Arg-101 to reach the pyrimidine bind ing pocket. From the 386-fold reduction in (k(cat)/K-m)(UpA) in RNase 4;R101N, it is concluded that this residue functions in uridine bindin g, analogous to Ser-123 in RNase A. In addition, it may have an effect on Asp-80. The 2-fold increase in (k(cat)/K-m)(CpA) in the mutant R10 1N and the close proximity of the side chains of Arg-101 and Asp-80 su ggested that the latter could be involved in suppressing CpA catalysis , The substrate specificity of RNase 4;D80A was completely reversed: ( k(cat)/K-m)(UpA) decreased 159-fold, whereas (k(cat)/K-m)(CpA) increas ed 233-fold. The effect on CpA was unexpected, because the correspondi ng residue in bovine pancreatic RNase A (Asp-83) hardly affects cytidi ne-containing substrates. Furthermore, the residue is conserved in nea rly all sequences of mammalian RNase 1. Thus, an evolutionary highly c onserved residue does not necessarily function in the same way in homo logous enzymes. A model, which proposes that the structure of RNase 4 has been optimized to permanently fix the position of Asp-80 and imped e its movement away from the pyrimidine binding pocket, is presented.