FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
The Biochemical journal1975; 149(3); 627-635; doi: 10.1042/bj1490627

Horse liver alcohol dehydrogenase. A study of the essential lysine residue.

Abstract: 1. The inactivation of horse liver alcohol dehydrogenase by pyridoxal 5'-phosphate in phosphate buffer, pH8, at 10 degrees C was investigated. Activity declines to a minimum value determined by the pyridoxal 5'-phosphate concentration. The maximum inactivation in a single treatment is 75%. This limit appears to be set by the ratio of the first-order rate constants for interconversion of inactive covalently modified enzyme and a readily dissociable non-covalent enzyme-modifier complex. 2. Reactivation was virtually complete on 150-fold dilution: first-order analysis yielded an estimate of the rate constant (0.164min-1), which was then used in the kinetic analysis of the forward inactivation reaction. This provided estimates for the rate constant for conversion of non-covalent complex into inactive enzyme (0.465 min-1) and the dissociation constant of the non-covalent complex (2.8 mM). From the two first-order constants, the minimum attainable activity in a single cycle of treatment may be calculated as 24.5%, very close to the observed value. 3. Successive cycles of modification followed by reduction with NaBH4 each decreased activity by the same fraction, so that three cycles with 3.6 mM-pyridoxal 5'-phosphate decreased specific activity to about 1% of the original value. The absorption spectrum of the enzyme thus treated indicated incorporation of 2-3 mol of pyridoxal 5'-phosphate per mol of subunit, covalently bonded to lysine residues. 4. NAD+ and NADH protected the enzyme completely against inactivation by pyridoxal 5'-phosphate, but ethanol and acetaldehyde were without effect. 5. Pyridoxal 5'-phosphate used as an inhibitor in steady-state experiments, rather than as an inactivator, was non-competitive with respect to both NADH and acetaldehyde. 6. The partially modified enzyme (74% inactive) showed unaltered apparent Km values for NAD+ and ethanol, indicating that modified enzyme is completely inactive, and that the residual activity is due to enzyme that has not been covalently modified. 7. Activation by methylation with formaldehyde was confirmed, but this treatment does not prevent subsequent inactivation with pyridoxal 5'-phosphate. Presumably different lysine residues are involved. 8. It is likely that the essential lysine residue modified by pyridoxal 5'-phosphate is involved either in binding the coenzymes or in the catalytic step. 9. Less detailed studies of yeast alcohol dehydrogenase suggest that this enzyme also possesses an essential lysine residue.
Get Full Text
Publication Date: 1975-09-01 PubMed ID: 173294PubMed Central: PMC1165669DOI: 10.1042/bj1490627Google 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.
LinkedInCopy
  • Journal Article

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.

The research focuses on understanding how the enzyme horse liver alcohol dehydrogenase is inactivated by a compound called pyridoxal 5′-phosphate. It also explores how this modification affects the enzyme’s functionality and the role of an essential amino acid, lysine, in this process.

Study and Findings Overview

  • The research begins by investigating the inactivation of horse liver alcohol dehydrogenase by pyridoxal 5′-phosphate in a phosphate buffer with pH8, at 10°C. It was observed that the activity of the enzyme decreased to a minimum level that was determined by the concentration of Pyridoxal 5′-phosphate.
  • It was found that the maximum inactivation possible in a single treatment was 75% and could be determined by the ratio of the first-order rate constants for changing the inactive, covalently modified enzyme, and a non-covalent enzyme-modifier complex that could easily separate.
  • The enzyme was mostly reactivated on dilution, estimating the rate constant for this reactivation. This rate constant was then used in the analysis of the forward inactivation reaction.

Effects and Implications of Modification Cycles

  • The research then looked into the effects of successive cycles of modification and reduction on the enzyme, each decreasing the activity by the same fraction. Therefore, three cycles using Pyridoxal 5′-phosphate decreased the enzyme’s specific activity to about 1% of the original value.
  • It was found that NAD+ and NADH protected the enzyme against inactivation by Pyridoxal 5′-phosphate. The partially inactivated enzyme showed no changes in apparent Km values for NAD+ and ethanol, confirming that the modified enzyme was completely inactivated, leaving the residual activity to unmodified enzyme.

Role of Lysine Residue and Further Studies

  • This enzyme modification was attributed to the pyridoxal 5′-phosphate being covalently bonded to lysine residues. The essential lysine residue that is modified was implicated either in binding the coenzymes or in the catalytic step.
  • The study also suggested that a similar pattern might be followed by yeast alcohol dehydrogenase, indicating that this enzyme also might have an essential lysine residue. However, these claims were based on less detailed studies and would need further investigation to be conclusive.

In summary, this research provides an in-depth analysis of the inactivation process of the horse liver alcohol dehydrogenase enzyme by Pyridoxal 5′-phosphate and the role of lysine residues in this process. It also offers insights that can be important for understanding similar enzymes and processes in different species and contexts.

Cite This Article

APA
Chen SS, Engel PC. (1975). Horse liver alcohol dehydrogenase. A study of the essential lysine residue. Biochem J, 149(3), 627-635. https://doi.org/10.1042/bj1490627

Publication

The Biochemical journal
ISSN: 0264-6021
NlmUniqueID: 2984726R
Country: England
Language: English
Volume: 149
Issue: 3
Pages: 627-635

Researcher Affiliations

Chen, S S
    Engel, P C

      MeSH Terms

      • Alcohol Oxidoreductases / antagonists & inhibitors
      • Alcohol Oxidoreductases / metabolism
      • Animals
      • Horses
      • In Vitro Techniques
      • Kinetics
      • Liver / enzymology
      • Lysine / metabolism
      • Models, Chemical
      • NAD
      • Pyridoxal Phosphate / pharmacology
      • Time Factors

      References

      This article includes 23 references
      1. Dworschack R, Tarr G, Plapp BV. Identification of the lysine residue modified during the activation of acetimidylation of horse liver alcohol dehydrogenase.. Biochemistry 1975 Jan 28;14(2):200-3.
        pubmed: 1168062doi: 10.1021/bi00673a002google scholar: lookup
      2. Engel PC, Dalziel K. Kinetic studies of glutamate dehydrogenase. The reductive amination of 2-oxoglutarate.. Biochem J 1970 Jul;118(3):409-19.
        pubmed: 4394334doi: 10.1042/bj1180409google scholar: lookup
      3. Chen SS, Engel PC. The equilibrium position of the reaction of bovine liver glutamate dehydrogenase with pyridoxal5'-phosphate. A demonstration that covalent modification with this reagent completely abolishes catalytic activity.. Biochem J 1975 May;147(2):351-8.
        pubmed: 1237292doi: 10.1042/bj1470351google scholar: lookup
      4. Chen SS, Engel PC. Modification of pig M4 lactate dehydrogenase by pyridoxal 5'-phosphate. Demonstration of an essential lysine residue.. Biochem J 1975 Jul;149(1):107-13.
        pubmed: 1238085doi: 10.1042/bj1490107google scholar: lookup
      5. DALZIEL K. Kinetic studies of liver alcohol dehydrogenase.. Biochem J 1962 Aug;84(2):244-54.
        pubmed: 13883267doi: 10.1042/bj0840244google scholar: lookup
      6. KITZ R, WILSON IB. Esters of methanesulfonic acid as irreversible inhibitors of acetylcholinesterase.. J Biol Chem 1962 Oct;237:3245-9.
        pubmed: 14033211
      7. Piszkiewicz D, Smith EL. Bovine liver glutamate dehydrogenase. Equilibria and kinetics of inactivation by pyridoxal.. Biochemistry 1971 Nov 23;10(24):4538-44.
        pubmed: 4401128doi: 10.1021/bi00800a030google scholar: lookup
      8. Eklund H, Nordström B, Zeppezauer E, Söderlund G, Ohlsson I, Boiwe T, Brändén CI. The structure of horse liver alcohol dehydrogenase.. FEBS Lett 1974 Aug 25;44(2):200-4.
        pubmed: 4473096doi: 10.1016/0014-5793(74)80725-8google scholar: lookup
      9. Jörnvall H. Partial similarities between yeast and liver alcohol dehydrogenases.. Proc Natl Acad Sci U S A 1973 Aug;70(8):2295-8.
        pubmed: 4599620doi: 10.1073/pnas.70.8.2295google scholar: lookup
      10. Johnson GS, Deal WC Jr. Inactivation of tetrameric rabbit muscle pyruvate kinase by specific binding of 2 to 4 moles of pyridoxal 5'-phosphate.. J Biol Chem 1970 Jan 25;245(2):238-45.
        pubmed: 5460887
      11. Pietruszko R, Theorell H. Subunit composition of horse liver alcohol dehydrogenase.. Arch Biochem Biophys 1969 Apr;131(1):288-98.
        pubmed: 5813834doi: 10.1016/0003-9861(69)90133-7google scholar: lookup
      12. Ronchi S, Zapponi MC, Ferri G. Inhibition by pyridoxal-phosphate of glyceraldehyde-3-phosphate dehydrogenase.. Eur J Biochem 1969 Apr;8(3):325-31.
        pubmed: 5816756doi: 10.1111/j.1432-1033.1969.tb00531.xgoogle scholar: lookup
      13. DALZIEL K. KINETIC STUDIES OF LIVER ALCOHOL DEHYDROGENASE AND PH EFFECTS WITH COENZYME PREPARATIONS OF HIGH PURITY.. J Biol Chem 1963 Aug;238:2850-8.
        pubmed: 14063314
      14. Chen SS, Engel PC. Inactivation of nicotinamide--adenine dinucleotide-linked dehydrogenases by pyridoxal 5'-phosphate.. Biochem Soc Trans 1975;3(1):80-2.
        pubmed: 165109doi: 10.1042/bst0030080google scholar: lookup
      15. Silverstein E, Sulebele G. Equilibrium kinetic study of the catalytic mechanism of bovine liver glutamate dehydrogenase.. Biochemistry 1973 May 22;12(11):2164-72.
        pubmed: 4145192doi: 10.1021/bi00735a024google scholar: lookup
      16. Plapp BV. Enhancement of the activity of horse liver alcohol dehydrogenase by modification of amino groups at the active sites.. J Biol Chem 1970 Apr 10;245(7):1727-35.
        pubmed: 4314596
      17. Goldin BR, Frieden C. The effect of pyridoxal phosphate modification on the catalytic and regulatory properties of bovine liver glutamate dehydrogenase.. J Biol Chem 1972 Apr 10;247(7):2139-44.
        pubmed: 4335864
      18. McKinley-McKee JS, Morris DL. The lysines in liver alcohol dehydrogenase. Chemical modification with pyridoxal 5'-phosphate and methyl picolinimidate.. Eur J Biochem 1972 Jun 23;28(1):1-11.
        pubmed: 4340477doi: 10.1111/j.1432-1033.1972.tb01877.xgoogle scholar: lookup
      19. Jörnvall H. Reactive lysine residues in horse liver alcohol dehydrogenase.. Biochem Biophys Res Commun 1973 Apr 16;51(4):1069-76.
        pubmed: 4349993doi: 10.1016/0006-291x(73)90036-3google scholar: lookup
      20. Tsai CS, Tsai YH, Lauzon G, Cheng ST. Structure and activity of methylated horse liver alcohol dehydrogenase.. Biochemistry 1974 Jan 29;13(3):440-3.
        pubmed: 4358947doi: 10.1021/bi00700a007google scholar: lookup
      21. Dunn MF. A comparison of the kinetics and stoichiometry of proton uptake with aldehyde reduction for liver alcohol dehydrogenase under single turnover conditions.. Biochemistry 1974 Mar 12;13(6):1146-51.
        pubmed: 4360779doi: 10.1021/bi00703a015google scholar: lookup
      22. Chen S, Engel PC. Protection of glutamate dehydrogenase by nicotinamide-adenine dinucleotide against reversible inactivation by pyridoxal 5'-phosphate as a sensitive indicator of conformational change induced by substrates and substrate analogues.. Biochem J 1974 Dec;143(3):569-74.
        pubmed: 4376949doi: 10.1042/bj1430569google scholar: lookup
      23. Sogin DC, Plapp BV. Activation and inactivation of horse liver alcohol dehydrogenase with pyridoxal compounds.. J Biol Chem 1975 Jan 10;250(1):205-10.
        pubmed: 1170167

      Citations

      This article has been cited 9 times.
      1. González-Nilo FD, Vega R, Cardemil E. Molecular modeling of the complexes between Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase and the ATP analogs pyridoxal 5'-diphosphoadenosine and pyridoxal 5'-triphosphoadenosine. Specific labeling of lysine 290. J Protein Chem 2000 Jan;19(1):67-73.
        doi: 10.1023/a:1007099010762pubmed: 10882174google scholar: lookup
      2. Dickinson FM. The purification and some properties of the Mg(2+)-activated cytosolic aldehyde dehydrogenase of Saccharomyces cerevisiae. Biochem J 1996 Apr 15;315 ( Pt 2)(Pt 2):393-9.
        doi: 10.1042/bj3150393pubmed: 8615805google scholar: lookup
      3. Martín J, Mancheño JM, Arche R. Inactivation of penicillin acylase from Kluyvera citrophila by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline: a case of time-dependent non-covalent enzyme inhibition. Biochem J 1993 May 1;291 ( Pt 3)(Pt 3):907-14.
        doi: 10.1042/bj2910907pubmed: 8489517google scholar: lookup
      4. Gould KG, Engel PC. Modification of mouse testicular lactate dehydrogenase by pyridoxal 5'-phosphate. Biochem J 1980 Nov 1;191(2):365-71.
        doi: 10.1042/bj1910365pubmed: 6786279google scholar: lookup
      5. Hart GJ, Leeper FJ, Battersby AR. Modification of hydroxymethylbilane synthase (porphobilinogen deaminase) by pyridoxal 5'-phosphate. Demonstration of an essential lysine residue. Biochem J 1984 Aug 15;222(1):93-102.
        doi: 10.1042/bj2220093pubmed: 6433896google scholar: lookup
      6. Rakitzis ET. Kinetic analysis of protein modification reactions at equilibrium. Biochem J 1989 Nov 1;263(3):855-9.
        doi: 10.1042/bj2630855pubmed: 2597131google scholar: lookup
      7. Rakitzis ET. Kinetic analysis of regeneration by dilution of a covalently modified protein. Biochem J 1990 Jun 15;268(3):669-70.
        doi: 10.1042/bj2680669pubmed: 2363704google scholar: lookup
      8. Tsai CS. Kinetic and mechanistic studies of methylated liver alcohol dehydrogenase. Biochem J 1978 Aug 1;173(2):483-96.
        doi: 10.1042/bj1730483pubmed: 697732google scholar: lookup
      9. Chen SS, Engel PC. Dogfish M4 lactate dehydrogenase: reversible inactivation by pyridoxal 5'-phosphate and complete protection in complexes that mimic the active ternary complex. Biochem J 1975 Nov;151(2):447-9.
        doi: 10.1042/bj1510447pubmed: 175780google scholar: lookup
      Subscribe to our newsletter
      Join 99,850 horse owners like you who receive our email updates on horse health and nutrition.