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.

摘要: 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 vitro substrate for NAD(P)H-dependent reduction. The catalytic roles conserved active-site residues (Tyr 51 , Lys 80 His 113 ) conversion reactive α-dicarbonyl moiety are not well understood. Using wild-type mutated individually replaced by alanine) forms AKR2B5, we have conducted steady-state transient kinetic studies effects varied pH deuterium isotopic substitutions coenzyme solvent on enzymatic rates Each mutation caused 10 3 –10 4 -fold decrease rate constant hydride transfer from NADH to PQ, whose value enzyme was determined ∼8×10 2  s −1 . data presented support an enzymic mechanism which proton bridge protonated side chain (p K =8.6±0.1) group adjacent acceptor facilitates chemical reaction step. contributes positioning catalysis. Contrasting its role general acid xylose, Tyr controls release hydroquinone product. proposed chemistry action involves delivery both hydrogens required reduction undergoing (stereoselective) transformation. Hydride probably precedes p 7.3±0.3 NAD + -bound appears suitable protonation anion =8.8). These results show that is unusually plastic exploitation residues, assistance provided α-dicarbonyls differs utilized xylose.