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Home / Equine Research Bank / Biochem J / Horse liver alcohol dehydrogenase. A study of the essential ...
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.
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Publication Date: 1975-09-01 PubMed ID: 173294PubMed Central: PMC1165669DOI: 10.1042/bj1490627Google Scholar: Lookup
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  • 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

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