Survey of catalytic residues and essential roles of glutamate-alpha170 and aspartate-alpha335 in coenzyme B12-dependent diol dehydratase.
The importance of each active-site residue in adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca was estimated using mutant enzymes in which one of the residues interacting with substrate and/or K(+) was mutated to Ala or another amino acid residue. The Ealpha170A and Dalpha335A mutants were totally inactive, and the Halpha143A mutant showed only a trace of activity, indicating that Glu-alpha170, Asp-alpha335, and His-alpha143 are catalytic residues. The Qalpha141A, Qalpha296A, and Salpha362A mutants showed partial activity. It was suggested from kinetic parameters that Gln-alpha296 is important for substrate binding and Gln-alpha296 and Gln-alpha141 for preventing the enzyme from mechanism-based inactivation. The Ealpha221A, Ealpha170H, and Dalpha335A did not form the (alphabetagamma)(2) complex, suggesting that these mutations indirectly disrupt subunit contacts. Among other Glu-alpha170 and Asp-alpha335 mutants, Ealpha170D and Ealpha170Q were 2.2 +/- 0.3% and 0.02% as active as the wild-type enzyme, respectively, whereas Dalpha335N was totally inactive. Kinetic analysis indicated that the presence and the position of a carboxyl group in the residue alpha170 are essential for catalysis as well as for the continuous progress of catalytic cycles. It was suggested that the roles of Glu-alpha170 and Asp-alpha335 are to participate in the binding of substrate and intermediates and keep them appropriately oriented and to function as a base in the dehydration of the 1,1-diol intermediate. In addition, Glu-alpha170 seems to stabilize the transition state for the hydroxyl group migration from C2 to C1 by accepting the proton of the spectator hydroxyl group on C1.
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-naka, Okayama 700-8530, Japan.
Masahiro
M
Kawata
Koichiro
K
Kinoshita
Sumihisa
S
Takahashi
Ken-ichi
K
Ogura
Noriaki
N
Komoto
Mamoru
M
Yamanishi
Takamasa
T
Tobimatsu
Tetsuo
T
Toraya
D001224:Aspartic Acid; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D003038:Cobamides / Q000378:metabolism; D018698:Glutamic Acid; D007700:Kinetics; D041121:Klebsiella oxytoca / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D011408:Propanediol Dehydratase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001426:Bacterial Proteins; D003038:Cobamides; D001224:Aspartic Acid; D018698:Glutamic Acid; D011408:Propanediol Dehydratase; C000913:cobamamide