Selective alteration of substrate specificity by replacement of aspartic acid-189 with lysine in the binding pocket of trypsin.
To test the role of Asp-189 which is located at the base of the substrate binding pocket in determining the specificity of trypsin toward basic substrates, this residue was replaced with a lysine residue by site-directed mutagenesis. Both rat trypsinogen and Lys-189 trypsinogen were expressed and secreted into the periplasmic space of Escherichia coli. The proteins were purified to homogeneity and activated by porcine enterokinase, and their catalytic activities were determined on natural and synthetic substrates. Lys-189 trypsin displayed no catalytic activity toward arginyl and lysyl substrates. Further, there was no compensatory change in specificity toward acidic substrates; no cleavage of aspartyl or glutamyl bonds was detected. Additional studies of substrate specificity involving gas-phase sequence analyses of digested natural substrates revealed an inherent but low chymotrypsin-like activity of trypsin. This activity was retained but modified by the Asp to Lys change at position 189. In addition to hydrolyzing phenylalanyl and tyrosyl peptide bonds, the mutant enzyme has the unique property of cleaving leucyl bonds. On the basis of computer graphic modeling studies of the Lys-189 side chain, it appears that the positively charged NH2 group is directed outside the substrate binding pocket. The resulting hydrophobic cavity may explain the altered substrate specificity of the mutant enzyme. The relatively low chymotrypsin-like activity of both recombinant enzymes may be due to distorted positioning of the scissile bond with respect to the catalytic triad rather than to the lack of sufficient interaction between the hydrophobic side chains and the substrate binding pocket of the enzyme.
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D003196:Computer Graphics; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008239:Lysine; D008958:Models, Molecular; D010957:Plasmids; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014357:Trypsin / Q000235:genetics* / Q000378:metabolism; D014362:Trypsinogen / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D001224:Aspartic Acid; D014362:Trypsinogen; D014357:Trypsin; D008239:Lysine