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Home / Equine Research Bank / Biochemistry / Mechanism of binding of horse liver alcohol dehydrogenase an...
Biochemistry1988; 27(14); 5082-5088; doi: 10.1021/bi00414a020

Mechanism of binding of horse liver alcohol dehydrogenase and nicotinamide adenine dinucleotide.

Abstract: The binding of NAD+ to liver alcohol dehydrogenase was studied by stopped-flow techniques in the pH range from 6.1 to 10.9 at 25 degrees C. Varying the concentrations of NAD+ and a substrate analogue used to trap the enzyme-NAD+ complex gave saturation kinetics. The same maximum rate constants were obtained with or without the trapping agent and by following the reaction with protein fluorescence or absorbance of a ternary complex. The data fit a mechanism with diffusion-controlled association of enzyme and NAD+, followed by an isomerization with a forward rate constant of 500 s-1 at pH 8: E E-NAD+ *E-NAD+. The isomerization may be related to the conformational change determined by X-ray crystallography of free enzyme and enzyme-coenzyme complexes. Overall bimolecular rate constants for NAD+ binding show a bell-shaped pH dependence with apparent pK values at 6.9 and 9.0. Acetimidylation of epsilon-amino groups shifts the upper pK to a value of 11 or higher, suggesting that Lys-228 is responsible for the pK of 9.0. Formation of the enzyme-imidazole complex abolishes the pK value of 6.9, suggesting that a hydrogen-bonded system extending from the zinc-bound water to His-51 is responsible for this pK value. The rates of isomerization of E-NAD+ and of pyrazole binding were maximal at pH below a pK of about 8, which is attributable to the hydrogen-bonded system. Acetimidylation of lysines or displacement of zinc-water with imidazole had little effect on the rate of isomerization of the E-NAD+ complex.(ABSTRACT TRUNCATED AT 250 WORDS)
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Publication Date: 1988-07-12 PubMed ID: 3167032DOI: 10.1021/bi00414a020Google Scholar: Lookup
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  • Journal Article
  • 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 study explores the process and mechanisms behind the binding of nicotinamide adenine dinucleotide (NAD+) to horse liver alcohol dehydrogenase, a specific enzyme, by utilizing stopped-flow techniques in different pH conditions.

Methodology

  • The study employed stopped-flow techniques over a diverse pH range from 6.1 to 10.9 at 25 degrees Celsius.
  • The concentrations of NAD+ and a substrate analogue, purposed to trap the enzyme-NAD+ complex, were varied interventionally.

Research Findings

  • Data showed consistent maximum rate constants regardless of the presence of the trapping agent or whether the reaction was followed through protein fluorescence or absorbance of a ternary complex.
  • These findings fitted a mechanism that included diffusion-controlled association of the enzyme and NAD+, then followed an isomerization with a forward rate constant of 500 s-1 at pH 8.
  • The researchers proposed that this isomerization process could be related to the conformational alterations determined by X-ray crystallography on the free enzyme and enzyme-coenzyme complexes.

pH Dependence

  • The researchers observed that the bimolecular rate constants for NAD+ binding demonstrated a bell-shaped pH dependence with apparent pK values at 6.9 and 9.0.
  • The function of different enzyme components was analyzed, suggesting that Lys-228 could be responsible for the pK of 9.0 and a hydrogen-bonded system extending from the zinc-bound water to His-51 for the pK value of 6.9.
  • The rates of isomerization of E-NAD+ and of pyrazole binding were highest at a pH below a pK of about 8, which is attributable to the aforementioned hydrogen-bonded system. Acetimidylation of lysines and displacement of zinc-water with imidazole had minimal effect on the E-NAD+ complex’s rate of isomerization.

Conclusion

  • These findings provide valuable insights on how NAD+ binds to the alcohol dehydrogenase enzyme in horse liver.
  • Understanding this mechanism could contribute to innovative strategies related to enzymology and cell biology, potentially leading to novel therapeutics for diseases related to enzymatic disorders.

Cite This Article

APA
Sekhar VC, Plapp BV. (1988). Mechanism of binding of horse liver alcohol dehydrogenase and nicotinamide adenine dinucleotide. Biochemistry, 27(14), 5082-5088. https://doi.org/10.1021/bi00414a020

Publication

Biochemistry
ISSN: 0006-2960
NlmUniqueID: 0370623
Country: United States
Language: English
Volume: 27
Issue: 14
Pages: 5082-5088

Researcher Affiliations

Sekhar, V C
  • Department of Biochemistry, University of Iowa, Iowa City 52242.
Plapp, B V

    MeSH Terms

    • Alcohol Dehydrogenase / metabolism
    • Algorithms
    • Animals
    • Horses
    • Hydrogen-Ion Concentration
    • Kinetics
    • Liver / enzymology
    • NAD / metabolism
    • Protein Conformation

    Grant Funding

    • AA00279 / NIAAA NIH HHS

    Citations

    This article has been cited 9 times.
    1. 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.
      doi: 10.1107/S2059798322008361pubmed: 36189742google scholar: lookup
    2. 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.
      doi: 10.1016/j.cbi.2021.109650pubmed: 34529977google scholar: lookup
    3. 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.
      doi: 10.1021/acs.biochem.7b00446pubmed: 28640600google scholar: lookup
    4. Kim K, Plapp BV. Inversion of substrate stereoselectivity of horse liver alcohol dehydrogenase by substitutions of Ser-48 and Phe-93. Chem Biol Interact 2017 Oct 1;276:77-87.
      doi: 10.1016/j.cbi.2016.12.016pubmed: 28025168google scholar: lookup
    5. Plapp BV. Conformational changes and catalysis by alcohol dehydrogenase. Arch Biochem Biophys 2010 Jan 1;493(1):3-12.
      doi: 10.1016/j.abb.2009.07.001pubmed: 19583966google scholar: lookup
    6. Kazuoka T, Oikawa T, Muraoka I, Kuroda S, Soda K. A cold-active and thermostable alcohol dehydrogenase of a psychrotorelant from Antarctic seawater, Flavobacterium frigidimaris KUC-1. Extremophiles 2007 Mar;11(2):257-67.
      doi: 10.1007/s00792-006-0034-1pubmed: 17072683google scholar: lookup
    7. Zimmerle CT, Frieden C. Analysis of progress curves by simulations generated by numerical integration. Biochem J 1989 Mar 1;258(2):381-7.
      doi: 10.1042/bj2580381pubmed: 2705989google scholar: lookup
    8. 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.
      doi: 10.1016/j.cbi.2024.110992pubmed: 38579923google scholar: lookup
    9. 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.
      doi: 10.1016/j.cbi.2023.110558pubmed: 37247811google scholar: lookup
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