1306770-89-7Relevant articles and documents
Mechanistic evaluation of MelA α-galactosidase from citrobacter freundii: A family 4 glycosyl hydrolase in which oxidation is rate-limiting
Chakladar, Saswati,Cheng, Lydia,Choi, Mary,Liu, James,Bennet, Andrew J.
experimental part, p. 4298 - 4308 (2012/03/22)
The MelA gene from Citrobacter freundii, which encodes a glycosyl hydrolase family 4 (GH4) α-galactosidase, has been cloned and expressed in Escherichia coli. The recombinant enzyme catalyzes the hydrolysis of phenyl α-galactosides via a redox elimination-addition mechanism involving oxidation of the hydroxyl group at C-3 and elimination of phenol across the C-1-C-2 bond to give an enzyme-bound glycal intermediate. For optimal activity, the MelA enzyme requires two cofactors, NAD+ and Mn2+, and the addition of a reducing agent, such as mercaptoethanol. To delineate the mechanism of action for this GH4 enzyme, we measured leaving group effects, and the derived βlg values on V and V/K are indistinguishable from zero (-0.01 ± 0.02 and 0.02 ± 0.04, respectively). Deuterium kinetic isotope effects (KIEs) were measured for the weakly activated substrate phenyl α-d-galactopyranoside in which isotopic substitution was incorporated at C-1, C-2, or C-3. KIEs of 1.06 ± 0.07, 0.91 ± 0.04, and 1.02 ± 0.06 were measured on V for the 1-2H, 2- 2H, and 3-2H isotopic substrates, respectively. The corresponding values on V/K were 1.13 ± 0.07, 1.74 ± 0.06, and 1.74 ± 0.05, respectively. To determine if the KIEs report on a single step or on a virtual transition state, we measured KIEs using doubly deuterated substrates. The measured DV/K KIEs for MelA-catalyzed hydrolysis of phenyl α-d-galactopyranoside on the dideuterated substrates, DV/K(3-D)/(2-D,3-D) and DV/K (2-D)/(2-D,3-D), are 1.71 ± 0.12 and 1.71 ± 0.13, respectively. In addition, the corresponding values on V, DV (3-D)/(2-D,3-D) and DV(2-D)/(2-D,3-D), are 0.91 ± 0.06 and 1.01 ± 0.06, respectively. These observations are consistent with oxidation at C-3, which occurs via the transfer of a hydride to the on-board NAD+, being concerted with proton removal at C-2 and the fact that this step is the first irreversible step for the MelA α-galactosidase-catalyzed reactions of aryl substrates. In addition, the rate-limiting step for Vmax must come after this irreversible step in the reaction mechanism.
