Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1992 Oct 1;287(Pt 1):145–150. doi: 10.1042/bj2870145

Steady-state kinetic analysis of aldehyde dehydrogenase from human erythrocytes.

G T Henehan 1, K F Tipton 1
PMCID: PMC1133136  PMID: 1417767

Abstract

The steady-state kinetics of purified cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from human erythrocytes have been studied at 37 degrees C. Previous studies of the enzyme from several mammalian sources, which used a lower assay temperature, have been difficult to interpret because of the substrate activation by acetaldehyde which led to complex kinetic behaviour. At 37 degrees C the initial-rate data do not depart significantly from Michaelis-Menten kinetics. Studies of the variation of initial rates as a function of the concentrations of both substrates and studies of the inhibition by NADH were consistent with a sequential mechanism being followed. High-substrate inhibition by acetaldehyde was competitive with respect to NAD+. The enzyme was not inhibited by the product acetate and thus the results of these studies, although consistent with an ordered mechanism in which NAD+ was the first substrate to bind, were inconclusive. That such a mechanism was followed was confirmed by determination of the initial-rate behaviour in the presence of acetaldehyde and glycolaldehyde as alternative substrates. When the reciprocal of the initial rate of NADH formation was plotted against the acetaldehyde concentration at a series of fixed ratios between that substrate and glycolaldehyde, a linear 'mixed inhibition' pattern was obtained, confirming the mechanism to be ordered with NAD+ being the leading substrate and with kinetically significant ternary complex-formation.

Full text

PDF
145

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Agarwal D. P., Eckey R., Harada S., Goedde H. W. Basis of aldehyde dehydrogenase deficiency in Orientals: immunochemical studies. Alcohol. 1984 Mar-Apr;1(2):111–118. doi: 10.1016/0741-8329(84)90065-x. [DOI] [PubMed] [Google Scholar]
  2. Blackwell L. F., Bennett A. F., Crow K. E., Buckley P. D., Deady L. W. A two-site model for the esterase and dehydrogenase activities of sheep liver aldehyde dehydrogenase. Pharmacol Biochem Behav. 1983;18 (Suppl 1):83–87. doi: 10.1016/0091-3057(83)90151-x. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Cleland W. W. The statistical analysis of enzyme kinetic data. Adv Enzymol Relat Areas Mol Biol. 1967;29:1–32. doi: 10.1002/9780470122747.ch1. [DOI] [PubMed] [Google Scholar]
  5. Cornish-Bowden A. Abrupt transitions in kinetic plots: an artifact of plotting procedures. Biochem J. 1988 Feb 15;250(1):309–311. doi: 10.1042/bj2500309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dickinson F. M. Studies on the mechanism of sheep liver cytosolic aldehyde dehydrogenase. Biochem J. 1985 Jan 1;225(1):159–165. doi: 10.1042/bj2250159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Duncan R. J. Aldehyde dehydrogenase. An enzyme with two distinct catalytic activities at a single type of active site. Biochem J. 1985 Aug 15;230(1):261–267. doi: 10.1042/bj2300261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Duncan R. J., Tipton K. F. The kinetics of pig brain aldehyde dehydrogenase. Eur J Biochem. 1971 Oct 26;22(4):538–543. doi: 10.1111/j.1432-1033.1971.tb01574.x. [DOI] [PubMed] [Google Scholar]
  9. Eckfeldt J. H., Yonetani T. Kinetics and mechanism of the F1 isozyme of horse liver aldehyde dehydrogenase. Arch Biochem Biophys. 1976 Mar;173(1):273–281. doi: 10.1016/0003-9861(76)90260-5. [DOI] [PubMed] [Google Scholar]
  10. Harrington M. C., Henehan G. T., Tipton K. F. The roles of human aldehyde dehydrogenase isoenzymes in ethanol metabolism. Prog Clin Biol Res. 1987;232:111–125. [PubMed] [Google Scholar]
  11. Hart G. J., Dickinson F. M. Kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase. Biochem J. 1982 Jun 1;203(3):617–627. doi: 10.1042/bj2030617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Helander A., Tottmar O. Cellular distribution and properties of human blood aldehyde dehydrogenase. Alcohol Clin Exp Res. 1986 Jan-Feb;10(1):71–76. doi: 10.1111/j.1530-0277.1986.tb05618.x. [DOI] [PubMed] [Google Scholar]
  13. Henehan G. T., Tipton K. F. The effects of assay temperature on the complex kinetics of acetaldehyde oxidation by aldehyde dehydrogenase from human erythrocytes. Biochem Pharmacol. 1991 Aug 8;42(5):979–984. doi: 10.1016/0006-2952(91)90278-d. [DOI] [PubMed] [Google Scholar]
  14. Huang C. Y. Use of alternative substrates to probe multisubstrate enzyme mechanisms. Methods Enzymol. 1979;63:486–500. doi: 10.1016/0076-6879(79)63021-5. [DOI] [PubMed] [Google Scholar]
  15. Loomes K. M., Kitson T. M. Aldehyde dehydrogenase catalyses acetaldehyde formation from 4-nitrophenyl acetate and NADH. Biochem J. 1986 Sep 1;238(2):617–619. doi: 10.1042/bj2380617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. MacGibbon A. K., Blackwell L. F., Buckley P. D. Kinetics of sheep-liver cytoplasmic aldehyde dehydrogenase. Eur J Biochem. 1977 Jul 1;77(1):93–100. doi: 10.1111/j.1432-1033.1977.tb11645.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Vallari R. C., Pietruszko R. Kinetic mechanism of the human cytoplasmic aldehyde dehydrogenase E1. Arch Biochem Biophys. 1981 Nov;212(1):9–19. doi: 10.1016/0003-9861(81)90338-6. [DOI] [PubMed] [Google Scholar]
  18. WILKINSON G. N. Statistical estimations in enzyme kinetics. Biochem J. 1961 Aug;80:324–332. doi: 10.1042/bj0800324. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES