Identification of catalytic residues of pepstatin-insensitive carboxyl proteinases from prokaryotes by site-directed mutagenesis.
Pepstatin-insensitive carboxyl proteinases from Pseudomonas sp. (PCP) and Xanthomonas sp. (XCP) have no conserved catalytic residue sequences, -Asp*-Thr-Gly- (Asp is the catalytic residue) for aspartic proteinases. To identify the catalytic residues of PCP and XCP, we selected presumed catalytic residues based on their high sequence similarity, assuming that such significant sites as catalytic residues will be generally conserved. Several Ala mutants of Asp or Glu residues were constructed and analyzed. The D170A, E222A, and D328A mutants for PCP and XD79A, XD169A, and XD348A mutants for XCP were not converted to mature protein after activation, and no catalytic activity could be detected in these mutants. The specificity constants toward chromogenic substrate of the other PCP and XCP mutants, except for the D84A mutant of PCP, were similar to that of wild-type PCP or XCP. Coupled with the result of chemical modification (Ito, M., Narutaki, S., Uchida, K., and Oda, K. (1999) J. Biochem. (Tokyo) 125, 210-216), a pair of Asp residues (170 and 328) for PCP and a pair of Asp residues (169 and 348) for XCP were elucidated to be their catalytic residues, respectively. The Glu(222) residue in PCP or Asp(79) residue in XCP was excluded from the candidates as catalytic residues, since the corresponding mutant retained its original activity.
Oyama
H
H
Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto, 606-8585, Japan.
Abe
S
S
Ushiyama
S
S
Takahashi
S
S
Oda
K
K
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D020134:Catalytic Domain; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004926:Escherichia coli; D018698:Glutamic Acid; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010436:Pepstatins / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014974:Xanthomonas / Q000201:enzymology* / Q000235:genetics
D010436:Pepstatins; D011994:Recombinant Proteins; C014195:Streptomyces pepsin inhibitor; D001224:Aspartic Acid; D018698:Glutamic Acid; D016282:Aspartic Acid Endopeptidases; C031375:pepstatin