A SEARCH FOR SINGLE SUBSTITUTIONS THAT ELIMINATE ENZYMATIC FUNCTION IN A BACTERIAL RIBONUCLEASE

Exhaustive-Substitution studies, where many amino acid replacements ar e individually tested at all positions in a natural protein, have prov en to be very valuable in probing the relationship between sequence an d function. The broad picture that has emerged from studies of this so rt is one of functional tolerance of substitution. We have applied thi s approach to barnase, a 110-residue bacterial ribonuclease. Because t he selection system used to score barnase mutants as active or inactiv e detects activity down to a level that can be approached by nonenzyme catalysts, mutants that test inactive are essentially devoid of enzym atic function. Of the 109 barnase positions subjected to substitution, only 15 (14%) are vulnerable to this extreme level of inactivation, a nd only 2 could not be substituted without such inactivation. A total of 33 substitutions (amounting to 5% of the explored substitutions) we re found to render barnase wholly inactive. The profoundly disruptive effects of all of these inactivating substitutions appear to result fr om either (1) replacement of a side chain that is directly involved in substrate binding or catalysis, (2) replacement of a substantially bu ried side chain, (3) introduction of a proline residue, or (4) replace ment of a glycine residue. Although substitutions of these types are f unctionally tolerated more often than not, the system used here indica tes that only these sorts of substitution are capable of single-handed ly reducing catalytic function to, or nearly to, levels that can be ac hieved by nonenzyme catalysts.