The catalytic mechanism of NADH-dependent reduction of 9,10-phenanthrenequinone by Candida tenuis xylose reductase reveals plasticity in an aldo-keto reductase active site.
Despite their widely varying physiological functions in carbonyl metabolism, AKR2B5 (Candida tenuis xylose reductase) and many related enzymes of the aldo-keto reductase protein superfamily utilise PQ (9,10-phenanthrenequinone) as a common in vitro substrate for NAD(P)H-dependent reduction. The catalytic roles of the conserved active-site residues (Tyr51, Lys80 and His113) of AKR2B5 in the conversion of the reactive alpha-dicarbonyl moiety of PQ are not well understood. Using wild-type and mutated (Tyr51, Lys80 and His113 individually replaced by alanine) forms of AKR2B5, we have conducted steady-state and transient kinetic studies of the effects of varied pH and deuterium isotopic substitutions in coenzyme and solvent on the enzymatic rates of PQ reduction. Each mutation caused a 10(3)-10(4)-fold decrease in the rate constant for hydride transfer from NADH to PQ, whose value in the wild-type enzyme was determined as approximately 8 x 10(2) s(-1). The data presented support an enzymic mechanism in which a catalytic proton bridge from the protonated side chain of Lys80 (pK=8.6+/-0.1) to the carbonyl group adjacent to the hydride acceptor carbonyl facilitates the chemical reaction step. His113 contributes to positioning of the PQ substrate for catalysis. Contrasting its role as catalytic general acid for conversion of the physiological substrate xylose, Tyr51 controls release of the hydroquinone product. The proposed chemistry of AKR2B5 action involves delivery of both hydrogens required for reduction of the alpha-dicarbonyl substrate to the carbonyl group undergoing (stereoselective) transformation. Hydride transfer from NADH probably precedes the transfer of a proton from Tyr51 whose pK of 7.3+/-0.3 in the NAD+-bound enzyme appears suitable for protonation of a hydroquinone anion (pK=8.8). These results show that the mechanism of AKR2B5 is unusually plastic in the exploitation of the active-site residues, for the catalytic assistance provided to carbonyl group reduction in alpha-dicarbonyls differs from that utilized in the conversion of xylose.
Pival
Simone L
SL
Institute of Biotechnology and Biochemical Engineering, Petersgasse 12, Graz University of Technology, A-8010 Graz, Austria.
Klimacek
Mario
M
Nidetzky
Bernd
B
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D000449:Aldehyde Reductase / Q000378:metabolism*; D000074408:Aldo-Keto Reductases; D002175:Candida / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015394:Molecular Structure; D009243:NAD / Q000737:chemistry / Q000378:metabolism*; D010616:Phenanthrenes / Q000378:metabolism*
D010616:Phenanthrenes; D009243:NAD; C005399:9,10-phenanthrenequinone; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase