Redesign of catalytic center of an enzyme: aspartic to serine proteinase.
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/I300H/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 2.10mM, 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.
Tanaka
Takuji
T
Department of Applied Microbiology and Food Science, University of Saskatchewan, 51 Campus Drive, Saskatoon, Saskatchewan, Canada S7N 5A8. takuji.tanaka@usask.ca
Yada
Rickey Y
RY
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D003198:Computer Simulation; D015195:Drug Design; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010664:Phenylmethylsulfonyl Fluoride / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014357:Trypsin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D010664:Phenylmethylsulfonyl Fluoride; D012697:Serine Endopeptidases; D014357:Trypsin; D016282:Aspartic Acid Endopeptidases