A CLN2-related and thermostable serine-carboxyl proteinase, kumamolysin: cloning, expression, and identification of catalytic serine residue.
The gene encoding kumamolysin, a thermostable pepstatin-insensitive carboxyl proteinase, was cloned and expressed. (i) Kumamolysin was synthesized as a large precursor consisting of two regions: amino-terminal prepro (188 amino acids) and mature proteins (384 amino acids). (ii) The deduced amino acid sequence of the mature region exhibited high similarity to those of such bacterial pepstatin-insensitive enzymes as Pseudomonas carboxyl proteinase (PSCP; EC 3.4.23.37, identity = 37%), Xanthomonas carboxyl proteinase (XCP; EC 3.4.23.33, identity = 36%), and human CLN2 gene product (identity = 36%), which is related to a fatal neurodegenerative disease. (iii) The presumed catalytic triad, Glu78, Asp82, Ser278 [three-dimensional structure of PSCP: Wlodawer, A. et al. (2001) Nature Struct. Biol., 8, 442-446], was found to be conserved in the amino acid sequence of kumamolysin. (iv) Kumamolysin was inactivated by such aldehyde-type inhibitors as Ac-Ile-Pro-Phe-CHO (K(i) = 0.7 0.14 microM). In PSCP, it has been clarified that these inhibitors form a hemiacetal linkage with the catalytic serine residue and inactivate the enzyme. (v) Mutational analysis of the Ser278 residue revealed that the mutant lost both auto-processing activity and proteolytic activity. These results strongly suggest that kumamolysin has a unique catalytic triad consisting of Glu78, Asp82, and Ser278 residues, as previously observed for PSCP.
Oyama
Hiroshi
H
Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
Hamada
Takatoshi
T
Ogasawara
Shin
S
Uchida
Kenichi
K
Murao
Sawao
S
Beyer
Bret B
BB
Dunn
Ben M
BM
Oda
Kohei
K
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D015183:Restriction Mapping; D017385:Sequence Homology; D012694:Serine / Q000378:metabolism*; D013329:Structure-Activity Relationship; D000091346:Tripeptidyl-Peptidase 1
D011480:Protease Inhibitors; D011994:Recombinant Proteins; D000091346:Tripeptidyl-Peptidase 1; D012694:Serine; C000717267:TPP1 protein, human; D016282:Aspartic Acid Endopeptidases; C078894:kumamolysin