PMID- 25500266 OWN - NLM STAT- MEDLINE DCOM- 20150629 LR - 20181113 IS - 1872-7786 (Electronic) IS - 0009-2797 (Print) IS - 0009-2797 (Linking) VI - 234 DP - 2015 Jun 5 TI - The rate-determining steps of aldo-keto reductases (AKRs), a study on human steroid 5beta-reductase (AKR1D1). PG - 360-5 LID - S0009-2797(14)00382-2 [pii] LID - 10.1016/j.cbi.2014.12.004 [doi] AB - Aldo-keto reductases (AKRs) are an expanding family of NAD(P)(H)-dependent oxidoreductases that catalyze the reduction of either carbonyl groups or alpha,beta-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 5beta-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. CI - Copyright (c) 2014 Elsevier Ireland Ltd. All rights reserved. FAU - Chen, Mo AU - Chen M AD - Center of Excellence in Environmental Toxicology and Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA. FAU - Jin, Yi AU - Jin Y AD - Center of Excellence in Environmental Toxicology and Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA. FAU - Penning, Trevor M AU - Penning TM AD - 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. LA - eng GR - 1R01CA090744/CA/NCI NIH HHS/United States GR - F32 DK089827/DK/NIDDK NIH HHS/United States GR - F32-DK089827/DK/NIDDK NIH HHS/United States GR - 1R01-DK47015/DK/NIDDK NIH HHS/United States GR - P30-ES13508/ES/NIEHS NIH HHS/United States GR - P30 ES013508/ES/NIEHS NIH HHS/United States GR - R01 DK047015/DK/NIDDK NIH HHS/United States GR - R01 CA090744/CA/NCI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20141211 PL - Ireland TA - Chem Biol Interact JT - Chemico-biological interactions JID - 0227276 RN - 0 (Steroids) RN - EC 1.- (Oxidoreductases) RN - EC 1.1.1.- (Aldo-Keto Reductases) RN - EC 1.1.1.21 (Aldehyde Reductase) RN - EC 1.3.99.6 (3-oxo-5 beta-steroid delta 4-dehydrogenase) SB - IM MH - Aldehyde Reductase/*metabolism MH - Aldo-Keto Reductases MH - Binding Sites MH - Catalysis MH - Humans MH - Kinetics MH - Oxidoreductases/*metabolism MH - Steroids/*metabolism MH - Substrate Specificity PMC - PMC4414691 MID - NIHMS648519 OTO - NOTNLM OT - Bile-acids OT - Cofactor release OT - Hydride transfer OT - Pre-steady state kinetics OT - Steroid hormones COIS- 6. Conflict of interest statement The authors declare no conflicts of interest associated with this manuscript. EDAT- 2014/12/17 06:00 MHDA- 2015/06/30 06:00 PMCR- 2016/06/05 CRDT- 2014/12/16 06:00 PHST- 2014/10/02 00:00 [received] PHST- 2014/12/02 00:00 [accepted] PHST- 2014/12/16 06:00 [entrez] PHST- 2014/12/17 06:00 [pubmed] PHST- 2015/06/30 06:00 [medline] PHST- 2016/06/05 00:00 [pmc-release] AID - S0009-2797(14)00382-2 [pii] AID - 10.1016/j.cbi.2014.12.004 [doi] PST - ppublish SO - Chem Biol Interact. 2015 Jun 5;234:360-5. doi: 10.1016/j.cbi.2014.12.004. Epub 2014 Dec 11.