title: The rate-determining steps of aldo-keto reductases (AKRs), a study on human steroid 5β-reductase (AKR1D1).
abstract: Aldo-keto reductases (AKRs) are an expanding family of NAD(P)(H)-dependent oxidoreductases that catalyze the reduction of either carbonyl groups or α,β-unsaturated ketones on a variety of endogenous and exogenous substrates. The enzymes catalyze a sequential ordered bi-bi kinetic mechanism, in which cofactor is bound first and released last. Using human steroid 5β-reductase (AKR1D1) as a representative enzyme, the influence of substrate structure on the rate-limiting steps of AKR catalysis has been previously determined. The rate of the chemistry step was found to differ by two orders of magnitude when different steroid substrates were used in single turnover experiments with AKR1D1. This difference was reflected in multiple turnover experiments. C17-C21 steroid substrates exhibited a fast chemistry step followed by slow product release as suggested by "burst" phase kinetics. By contrast, C27 steroids have a slower chemical step that determines the rate of the reaction and "burst-phase" kinetics are no longer observed. Here we present single turnover kinetic experiments and find that they support the existence of two different binding poses for fast substrates due to their biphasic nature. We also re-interpret the loss of "burst-phase" kinetics in the multiple turnover experiments as due to long range effects of the steroid side-chain interacting with distal parts of the steroid pocket to perturb the reaction trajectory for hydride transfer and thus reduce kcat. The ability of steroid structure and hence binding pose to influence rate determination in steroid transforming AKRs is discussed as a general phenomenon.
authors.affiliation: Center of Excellence in Environmental Toxicology and Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA.
authors.forename: Mo
authors.initials: M
authors.lastname: Chen
authors.affiliation: Center of Excellence in Environmental Toxicology and Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Jin
authors.affiliation: Center of Excellence in Environmental Toxicology and Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA. Electronic address: penning@upenn.edu.
authors.forename: Trevor M
authors.initials: TM
authors.lastname: Penning
mesh_terms: D000449:Aldehyde Reductase / Q000378:metabolism*; D000074408:Aldo-Keto Reductases; D001665:Binding Sites; D002384:Catalysis; D006801:Humans; D007700:Kinetics; D010088:Oxidoreductases / Q000378:metabolism*; D013256:Steroids / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D013256:Steroids; D010088:Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase; C021936:3-oxo-5 beta-steroid delta 4-dehydrogenase
keywords: Bile-acids; Cofactor release; Hydride transfer; Pre-steady state kinetics; Steroid hormones