Rational design in genetic engineering: dream or reality? A priori predictions for thrombin and ribonuclease a mutants

Simple electrostatic potential calculations were made for thrombin and ribonuclease A in order to predict the effect of hypothetical point mutations on the catalytic rate, k_cat. The numerical results were scaled using experimental data for trypsin and subtilisin mutants. The decrease in k_cat in D102A and D102N mutants of thrombin was predicted to be five and four orders of magnitude as compared with the wild-type enzyme. Using the same scaling procedure, an increase in the catalytic rate of a factor of ten was predicted in the D121A mutant of ribonuclease A. On the other hand, in the K41A mutant of the same enzyme the estimated rate change was a decrease of two orders of magnitude. It was found that the effects of the mutations investigated depend almost exclusively on geometric factors. Accordingly, it is concluded that in all point mutants of serine proteases where the buried aspartate is replaced by alanine or asparagine, the rate decrease will vary between four and six orders of magnitude.