ENGINEERED METAL REGULATION OF TRYPSIN SPECIFICITY

Histidine substrate specificity has been engineered into trypsin by cr eating metal binding sites for Ni2+ and Zn2+ ions. The sites bridge th e substrate and enzyme on the leaving-group side of the scissile bond. Application of simple steric and geometric criteria to a crystallogra phically derived enzyme-substrate model suggested that histidine speci ficity at the P2' position might be acheived by a tridentate site invo lving amino acid residues 143 and 151 of trypsin. Trypsin N143H/E151H hydrolyzes a P2'-His-containing peptide (AGPYAHSS) exclusively in the presence of nickel or zinc with a high level of catalytic efficiency. Since cleavage following the tyrosine residue is normally highly disfa vored by trypsin, this result demonstrates that a metal cofactor can b e used to modulate specificity in a designed fashion. The same geometr ic criteria applied in the primary S1 binding pocket suggested that th e single-site mutation D189H might effect metal-dependent His specific ity in trypsin. However, kinetic and crystallographic analysis of this variant showed that the design was unsuccessful because His189 rotate s away from substrate causing a large perturbation in adjacent surface loops. This observation suggests that the reason specificity modifica tion at the trypsin S1 site requires extensive mutagenesis is because the pocket cannot deform locally to accommodate alternate P1 side chai ns. By taking advantage of the extended subsites, an alternate substra te specificity has been engineered into trypsin.