A critical role for highly conserved Glu(610) residue of oligopeptidase B from Trypanosoma brucei in thermal stability.
Oligopeptidase B from Trypanosoma brucei (Tb OPB) is a virulence factor and therapeutic target in African sleeping sickness. Three glutamic acid residues at positions 607, 609 and 610 of the catalytic domain are highly conserved in the OPB subfamily. In this study, the roles of Glu(607), Glu(609) and Glu(610) in Tb OPB were investigated by site-directed mutagenesis. A striking effect on k(cat)/K(m) was obtained following mutation of Glu(607) to glutamine. In contrast, the heat stability of Tb OPB decreased markedly following the single mutation of Glu(610) to glutamine, although this mutation had significantly less effect on catalytic properties compared with the Glu(607) mutation. Although no differences were found in the tertiary and secondary structures between wild-type (WT) OPB and the E610Q mutant prior to heat treatment, the E610Q mutant is inactivated more rapidly than WT OPB following heat treatment in a manner correlating with its attendant structural changes. Trypsin digestion showed that the boundary regions between the beta-propeller and catalytic domain of the E610Q mutant are unfolded with heat treatment. It is concluded that Glu(607) is essential for the catalytic activity of Tb OPB and that Glu(610) plays a critical role in stabilization rather than catalytic activity despite their close proximity.
Mohd Ismail
Nor Ismaliza
NI
Department of Biological Science and Technology, University of Tokushima Graduate School, 2-1 Minamijosanjima, Tokushima 770-8506, Japan.
Yuasa
Tsuyoshi
T
Yuasa
Keizo
K
Nambu
Yuko
Y
Nisimoto
Makoto
M
Goto
Masaki
M
Matsuki
Hitoshi
H
Inoue
Masahiro
M
Nagahama
Masami
M
Tsuji
Akihiko
A
D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000818:Animals; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D005934:Glucagon / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006358:Hot Temperature; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012492:Salts / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics; D014357:Trypsin / Q000378:metabolism; D014508:Urea / Q000494:pharmacology
D015800:Protozoan Proteins; D012492:Salts; D018698:Glutamic Acid; D014508:Urea; D000324:Adrenocorticotropic Hormone; D005934:Glucagon; D012697:Serine Endopeptidases; D014357:Trypsin; C090703:oligopeptidase B