Survey of Catalytic Residues and Essential Roles of Glutamate- alpha 170 and Aspartate- alpha 335 in Coenzyme B sub(12)-dependent Diol Dehydratase

• Published in: The Journal of biological chemistry Vol. 281; no. 27; pp. 18327 - 18334
• Main Authors: Kawata, Masahiro, Kinoshita, Koichiro, Takahashi, Sumihisa, Ogura, Ken-ichi, Komoto, Noriaki, Yamanishi, Mamoru, Tobimatsu, Takamasa, Toraya, Tetsuo
• Format: Journal Article
• Language: English
• Published: 07.07.2006
• Subjects: Klebsiella oxytoca
• ISSN: 0021-9258; 1083-351X

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 super(+) was mutated to Ala or another amino acid residue. The E alpha 170A and D alpha 335A mutants were totally inactive, and the H alpha 143A mutant showed only a trace of activity, indicating that Glu- alpha 170, Asp- alpha 335, and His- alpha 143 are catalytic residues. The Q alpha 141A, Q alpha 296A, and S alpha 362A mutants showed partial activity. It was suggested from kinetic parameters that Gln- alpha 296 is important for substrate binding and Gln- alpha 296 and Gln- alpha 141 for preventing the enzyme from mechanism-based inactivation. The E alpha 221A, E alpha 170H, and D alpha 335A did not form the ( alpha beta gamma ) sub(2) complex, suggesting that these mutations indirectly disrupt subunit contacts. Among other Glu- alpha 170 and Asp- alpha 335 mutants, E alpha 170D and E alpha 170Q were 2.2 plus or minus 0.3% and 0.02% as active as the wild-type enzyme, respectively, whereas D alpha 335N was totally inactive. Kinetic analysis indicated that the presence and the position of a carboxyl group in the residue alpha 170 are essential for catalysis as well as for the continuous progress of catalytic cycles. It was suggested that the roles of Glu- alpha 170 and Asp- alpha 335 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- alpha 170 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.