title: Mechanistic evaluation of MelA α-galactosidase from Citrobacter freundii: a family 4 glycosyl hydrolase in which oxidation is rate-limiting.
abstract: 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 Mn(2+), 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 β(lg) 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-(2)H, 2-(2)H, and 3-(2)H 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 (D)V/K KIEs for MelA-catalyzed hydrolysis of phenyl α-D-galactopyranoside on the dideuterated substrates, (D)V/K((3-D)/(2-D,3-D)) and (D)V/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, (D)V((3-D)/(2-D,3-D)) and (D)V((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 V(max) must come after this irreversible step in the reaction mechanism.
authors.affiliation: Department of Chemistry, Simon Fraser University , 8888 University Drive, Burnaby, British Columbia, Canada.
authors.forename: Saswati
authors.initials: S
authors.lastname: Chakladar
authors.forename: Lydia
authors.initials: L
authors.lastname: Cheng
authors.forename: Mary
authors.initials: M
authors.lastname: Choi
authors.forename: James
authors.initials: J
authors.lastname: Liu
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Bennet
mesh_terms: D002240:Carbohydrate Sequence; D016971:Citrobacter freundii / Q000201:enzymology* / Q000235:genetics; D003903:Deuterium / Q000737:chemistry; D005690:Galactose / Q000378:metabolism; D006017:Glycolipids / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D008553:Melibiose / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D010084:Oxidation-Reduction; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D027981:Symporters / Q000235:genetics; D000519:alpha-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D006017:Glycolipids; D006023:Glycoproteins; D027981:Symporters; C021548:melibiose permease; D008553:Melibiose; D003903:Deuterium; D000519:alpha-Galactosidase; D005690:Galactose