TRYPSIN SPECIFICITY INCREASED THROUGH SUBSTRATE-ASSISTED CATALYSIS

Histidine 57 of the catalytic triad of trypsin was replaced with alani ne to determine whether the resulting variant would be capable of subs trate-assisted catalysis [Carter, P., & Wells, J. A. (1987) Science 23 7, 394-9]. A 2.5-fold increase in k(cat)/K-m was observed on tri- or t etrapeptide substrates containing p-nitroanilide leaving groups and hi stidine at P2. In contrast, hydrolysis of peptide substrates extending from P6 to P6' is improved 70-300-fold by histidine in the P2 or P1' position. This preference creates new protease specificities for seque nces HR down arrow, R down arrow H, HK down arrow, and K down arrow H. The ability of histidine from either the P2 or the P1' position of su bstrate to participate in catalysis emphasizes the considerable variab ility of proteolytically active orientations which can be assumed by t he catalytic triad. Trypsin H57A is able to hydrolyze fully folded orn ithine decarboxylase with complete specificity at a site containing th e sequence HRH. Trypsin H57A was compared to enteropeptidase in its ab ility to cleave a propeptide from trypsinogen. Trypsin H57A cleaved th e propeptide of a variant trypsinogen containing an introduced FPVDDDH R cleavage site only 100-fold slower than enteropeptidase cleaved tryp sinogen. The selective cleavage of folded proteins suggests that tryps in H57A can be used for specific peptide and protein cleavage. The ext ension of substrate-assisted catalysis to the chymotrypsin family of p roteolytic enzymes indicates that it may be possible to apply this str ategy to a wide range of serine proteases and thereby develop various unique specificities for peptide and protein hydrolysis.