title: pH-rate profiles support a general base mechanism for galactokinase (Lactococcus lactis).
abstract: Galactokinase (GALK), a member the Leloir pathway for normal galactose metabolism, catalyzes the conversion of α-d-galactose to galactose-1-phosphate. For this investigation, we studied the kinetic mechanism and pH profiles of the enzyme from Lactococcus lactis. Our results show that the mechanism for its reaction is sequential in both directions. Mutant proteins D183A and D183N are inactive (< 10000 fold), supporting the role of Asp183 as a catalytic base that deprotonates the C-1 hydroxyl group of galactose. The pH-kcat profile of the forward reaction has a pKa of 6.9 ± 0.2 that likely is due to Asp183. The pH-k(cat)/K(Gal) profile of the reverse reaction further substantiates this role as it is lacking a key pKa required for a direct proton transfer mechanism. The R36A and R36N mutant proteins show over 100-fold lower activity than that for the wild-type enzyme, thus suggesting that Arg36 lowers the pKa of the C-1 hydroxyl to facilitate deprotonation.
authors.affiliation: Institute For Enzyme Research and Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53726, USA. lareinha@wisc.edu
authors.forename: Laurie A
authors.initials: LA
authors.lastname: Reinhardt
authors.forename: James B
authors.initials: JB
authors.lastname: Thoden
authors.forename: Greg S
authors.initials: GS
authors.lastname: Peters
authors.forename: Hazel M
authors.initials: HM
authors.lastname: Holden
authors.forename: W W
authors.initials: WW
authors.lastname: Cleland
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D005686:Galactokinase / Q000737:chemistry* / Q000235:genetics; D005690:Galactose / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology*; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction
chemical_list: D001426:Bacterial Proteins; D000255:Adenosine Triphosphate; D005686:Galactokinase; D005690:Galactose
keywords: Galactokinase; General base; Kinetics; Mechanism; pH-rate profile