Deprotonation of the horse liver alcohol dehydrogenase-NAD+ complex controls formation of the ternary complexes.
Abstract: Binding of NAD+ to wild-type horse liver alcohol dehydrogenase is strongly pH-dependent and is limited by a unimolecular step, which may be related to a conformational change of the enzyme-NAD+ complex. Deprotonation during binding of NAD+ and inhibitors that trap the enzyme-NAD+ complex was examined by transient kinetics with pH indicators, and formation of complexes was monitored by absorbance and protein fluorescence. Reactions with pyrazole and trifluoroethanol had biphasic proton release, whereas reaction with caprate showed proton release followed by proton uptake. Proton release (200-550 s(-1)) is a common step that precedes binding of all inhibitors. At all pH values studied, the rate constants for proton release or uptake matched those for formation of ternary complexes, and the most significant quenching of protein fluorescence (or perturbation of adenine absorbance at 280 nm) was observed for enzyme species involved in deprotonation steps. Kinetic simulations of the combined transient data for the multiple signals indicate that all inhibitors bind faster and tighter to the unprotonated enzyme-NAD+ complex, which has a pK of about 7.3. The results suggest that rate-limiting deprotonation of the enzyme-NAD+ complex is coupled to the conformational change and controls the formation of ternary complexes.
Publication Date: 2005-09-21 PubMed ID: 16171395DOI: 10.1021/bi050865vGoogle Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- N.I.H.
- Extramural
- Research Support
- U.S. Gov't
- P.H.S.
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
This research investigates how the interaction between NAD+ and the enzyme found in horse liver, alcohol dehydrogenase, is heavily influenced by pH levels, particularly through a process called deprotonation. The study further suggests that this deprotonation step is integral to the formation of complexes involving these two substances.
Key points of the research
- The research begins with an exploration of the relationship between NAD+ and the enzyme known as alcohol dehydrogenase, sourced from the livers of horses.
- The study found that binding of NAD+ to this enzyme was heavily dependent on pH levels, with the process being limited by a single, unimolecular step. This step is believed to involve a change in the structure of the enzyme-NAD+ complex.
Role of deprotonation
- Deprotonation during the binding of NAD+ and inhibitors was further examined through transient kinetics with pH indicators.
- The formation of the complexes was monitored through absorbance and protein fluorescence.
- Deprotonation was found to be a common step that precedes the binding of all inhibitors, with different reactions showing varied levels of proton release and uptake.
- At all pH levels studied, there was a matching rate of proton release or uptake to the formation rate of the ternary complexes.
Relation between deprotonation and complex formation
- The researchers observed that the most significant quenching of protein fluorescence (or perturbation of adenine absorbance at 280 nm) occurred for enzyme species involved in deprotonation steps.
- Kinetic simulations showed that all inhibitors bind faster and tighter to the unprotonated enzyme-NAD+ complex.
- Results suggest a pH of about 7.3 for the unprotonated enzyme-NAD+ complex.
- The conclusion drawn is that a rate-limiting deprotonation of the enzyme-NAD+ complex is tied to the conformational change, thereby controlling the formation of ternary complexes.
Implications of the research
- This process of deprotonation and pH-level dependence may have implications for understanding how different substances interact in a biological context.
- Understanding the binding process between enzymes and coenzymes may aid in the development of more effective drugs or treatments for various health conditions.
Cite This Article
APA
Kovaleva EG, Plapp BV.
(2005).
Deprotonation of the horse liver alcohol dehydrogenase-NAD+ complex controls formation of the ternary complexes.
Biochemistry, 44(38), 12797-12808.
https://doi.org/10.1021/bi050865v Publication
Researcher Affiliations
- Department of Biochemistry, The University of Iowa, Iowa City, Iowa 52242, USA.
MeSH Terms
- Alcohol Dehydrogenase / chemistry
- Alcohol Dehydrogenase / metabolism
- Animals
- Enzyme Inhibitors / chemistry
- Enzyme Inhibitors / metabolism
- Horses
- Hydrogen-Ion Concentration
- Kinetics
- Liver / enzymology
- NAD / chemistry
- NAD / metabolism
- Protons
Grant Funding
- AA00279 / NIAAA NIH HHS
Citations
This article has been cited 17 times.- Plapp BV, Gakhar L, Subramanian R. Dependence of crystallographic atomic displacement parameters on temperature (25-150 K) for complexes of horse liver alcohol dehydrogenase. Acta Crystallogr D Struct Biol 2022 Oct 1;78(Pt 10):1221-1234.
- Pal S, Plapp BV. The Thr45Gly substitution in yeast alcohol dehydrogenase substantially decreases catalysis, alters pH dependencies, and disrupts the proton relay system. Chem Biol Interact 2021 Nov 1;349:109650.
- Plapp BV, Subramanian R. Alternative binding modes in abortive NADH-alcohol complexes of horse liver alcohol dehydrogenase. Arch Biochem Biophys 2021 Apr 15;701:108825.
- Shanmuganatham KK, Wallace RS, Ting-I Lee A, Plapp BV. Contribution of buried distal amino acid residues in horse liver alcohol dehydrogenase to structure and catalysis. Protein Sci 2018 Mar;27(3):750-768.
- Plapp BV, Savarimuthu BR, Ferraro DJ, Rubach JK, Brown EN, Ramaswamy S. Horse Liver Alcohol Dehydrogenase: Zinc Coordination and Catalysis. Biochemistry 2017 Jul 18;56(28):3632-3646.
- Jun SY, Walker AM, Kim H, Ralph J, Vermerris W, Sattler SE, Kang C. The Enzyme Activity and Substrate Specificity of Two Major Cinnamyl Alcohol Dehydrogenases in Sorghum (Sorghum bicolor), SbCAD2 and SbCAD4. Plant Physiol 2017 Aug;174(4):2128-2145.
- Raj SB, Ramaswamy S, Plapp BV. Yeast alcohol dehydrogenase structure and catalysis. Biochemistry 2014 Sep 16;53(36):5791-803.
- Plapp BV, Ramaswamy S. Atomic-resolution structures of horse liver alcohol dehydrogenase with NAD(+) and fluoroalcohols define strained Michaelis complexes. Biochemistry 2012 May 15;51(19):4035-48.
- Klimacek M, Brunsteiner M, Nidetzky B. Dynamic mechanism of proton transfer in mannitol 2-dehydrogenase from Pseudomonas fluorescens: mobile GLU292 controls proton relay through a water channel that connects the active site with bulk solvent. J Biol Chem 2012 Feb 24;287(9):6655-67.
- Kang C, Hayes R, Sanchez EJ, Webb BN, Li Q, Hooper T, Nissen MS, Xun L. Furfural reduction mechanism of a zinc-dependent alcohol dehydrogenase from Cupriavidus necator JMP134. Mol Microbiol 2012 Jan;83(1):85-95.
- Nagel ZD, Klinman JP. Update 1 of: Tunneling and dynamics in enzymatic hydride transfer. Chem Rev 2010 Dec 8;110(12):PR41-67.
- Plapp BV. Conformational changes and catalysis by alcohol dehydrogenase. Arch Biochem Biophys 2010 Jan 1;493(1):3-12.
- Pal S, Park DH, Plapp BV. Activity of yeast alcohol dehydrogenases on benzyl alcohols and benzaldehydes: characterization of ADH1 from Saccharomyces carlsbergensis and transition state analysis. Chem Biol Interact 2009 Mar 16;178(1-3):16-23.
- Klimacek M, Hellmer H, Nidetzky B. Catalytic mechanism of Zn2+-dependent polyol dehydrogenases: kinetic comparison of sheep liver sorbitol dehydrogenase with wild-type and Glu154-->Cys forms of yeast xylitol dehydrogenase. Biochem J 2007 Jun 15;404(3):421-9.
- Jacobi T, Kratzer DA, Plapp BV. Substitution of both histidines in the active site of yeast alcohol dehydrogenase 1 exposes underlying pH dependencies. Chem Biol Interact 2024 May 1;394:110992.
- Plapp BV. Solvent isotope and mutagenesis studies on the proton relay system in yeast alcohol dehydrogenase 1. Chem Biol Interact 2024 Jan 25;388:110853.
- Plapp BV, Kratzer DA, Souhrada SK, Warth E, Jacobi T. Specific base catalysis by yeast alcohol dehydrogenase I with substitutions of histidine-48 by glutamate or serine residues in the proton relay system. Chem Biol Interact 2023 Sep 1;382:110558.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
