Paper Citing NAMD - Abstract
Tanaka, T.; Yada, R.Y.
Redesign of catalytic center of an enzyme: aspartic to serine proteinase
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 323:947-953, OCT 22 2004
In an attempt to convert an aspartic proteinase into another class of proteinase, the catalytic residues of porcine pepsin were substituted with the catalytic triad characteristic of a serine proteinase, using trypsin as the model. Computer modeling Suggested six possible sites within porcine pepsin sequence for the introduction of the catalytic triad. The six mutants of pepsin were subsequently constructed and examined for their catalytic activities. Among the six mutants, two Mutants, D32S/1300H/G302D (MutI) and D32G/S35H/Y75S/I120D (MutJ), showed peptide hydrolysis activities. In comparison to the original activity of pepsin, the kinetic constants of these mutants were very low with K-m values of 4.10 and 2.10 mM, and k(0) values of 22.2 and 18.0 min(-1). In the presence of PMSF, a serine proteinase inhibitor, the activities for these mutants were inhibited by 86.5% and 80.1%, respectively, indicating that the catalytic triad of the trypsin had been successfully introduced into porcine pepsin. (C) 2004 Elsevier Inc. All rights reserved.
