The controlled introduction of multiple negative charge at single amino acid sites in subtilisin Bacillus lentus

The use of methanethiosulfonates as thiol-specific modifying reagents in th e strategy of combined site-directed mutagenesis and chemical modification allows virtually unlimited opportunities for creating new protein surface e nvironments. As a consequence of our interest in electrostatic manipulation as a means of tailoring enzyme activity and specificity, we have adopted t his approach for the controlled incorporation of multiple negative charges at single sites in the representative serine protease, subtilisin Bacillus lentus (SBL). A series of mono-, di- and triacidic acid methanethiosulfonat es were synthesized and used to modify cysteine mutants of SBL at positions 62 in the S-2 site, 156 and 166 in the S-1 site and 217 in the S-1' site. Kinetic parameters for these chemically modified mutant (CMM) enzymes were determined at pH 8.6 under conditions which ensured complete ionization of the unnatural amino acid side-chains introduced. The presence of up to thre e negative charges in the S-1, S-1' and S-2 subsites of SBL resulted in up to 11-fold lowered activity, possibly due to interference with oxyanion sta bilization of the transition state of the hydrolytic reactions catalyzed. E ach unit increase in negative charge resulted in a raising of K-M and a red uction of k(cat). However, no upper limit was observed for increases in K-M , whereas decreases in k(cat) reached a limiting value. Comparison with ste rically similar but uncharged CMMs revealed that electrostatic effects of n egative charges at positions 62, 156 and 217 are detrimental, but are benef icial at position 166. These results indicate that the ground-state binding of SBL to the standard substrate, Suc-AAPF-pNA, to SBL is reduced, but wit hout drastic attenuation of catalytic efficiency, and show that SBL tolerat es high levels of charge at single sites. (C) 1999 Elsevier Science Ltd. Al l rights reserved.