Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1992 Sep;1(9):1144–1153. doi: 10.1002/pro.5560010908

Avian 3-hydroxy-3-methylglutaryl-CoA lyase: sensitivity of enzyme activity to thiol/disulfide exchange and identification of proximal reactive cysteines.

P W Hruz 1, H M Miziorko 1
PMCID: PMC2142181  PMID: 1304393

Abstract

Catalysis by purified avian 3-hydroxy-3-methylglutaryl-CoA lyase is critically dependent on the reduction state of the enzyme, with less than 1% of optimal activity being observed with the air-oxidized enzyme. The enzyme is irreversibly inactivated by sulfhydryl-directed reagents with the rate of this inactivation being highly dependent upon the redox state of a critical cysteine. Methylation of reduced avian lyase with 1 mM 4-methylnitrobenzene sulfonate results in rapid inactivation of the enzyme with a k(inact) of 0.178 min-1. The oxidized enzyme is inactivated at a sixfold slower rate (k(inact) = 0.028 min-1). Inactivation of the enzyme with the reactive substrate analog 2-butynoyl-CoA shows a similar dependence upon the enzyme's redox state, with a sevenfold difference in k(inact) observed with oxidized vs. reduced forms of the enzyme. Chemical cross-linking of the reduced enzyme with stoichiometric amounts of the bifunctional reagents 1,3-dibromo-2-propanone (DBP) or N,N'-ortho-phenylene-dimaleimide (PDM) coincides with rapid inactivation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of enzyme treated with bifunctional reagent reveals a band of twice the molecular weight of the lyase monomer, indicating that an intersubunit cross-link has been formed. Differential labeling of native and cross-linked protein with [1-14C]iodoacetate has identified as the primary cross-linking target a cysteine within the sequence VSQAACR, which maps at the carboxy-terminus of the cDNA-deduced sequence of the avian enzyme (Mitchell, G.A., et al., 1991, Am. J. Hum. Genet. 49, 101). In contrast, bacterial HMG-CoA lyase, which contains no corresponding cysteine, is not cross-linked by comparable treatment with bifunctional reagent. These results provide evidence for a potential regulatory mechanism for the eukaryotic enzyme via thiol/disulfide exchange and identify a cysteinyl residue with the reactivity and juxtaposition required for participation in disulfide formation.

Full Text

The Full Text of this article is available as a PDF (2.6 MB).

Selected References

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

  1. BACHHAWAT B. K., ROBINSON W. G., COON M. J. The enzymatic cleavage of beta-hydroxy-beta-methylglutaryl coenzyme A to acetoacetate and acetyl coenzyme A. J Biol Chem. 1955 Oct;216(2):727–736. [PubMed] [Google Scholar]
  2. Bernert J. T., Jr, Sprecher H. An analysis of partial reactions in the overall chain elongation of saturated and unsaturated fatty acids by rat liver microsomes. J Biol Chem. 1977 Oct 10;252(19):6736–6744. [PubMed] [Google Scholar]
  3. Faull K., Bolton P., Halpern B., Hammond J., Danks D. M., Hähnel R., Wilkinson S. P., Wysocki S. J., Masters P. L. Letter: Patient with defect in leucine metabolism. N Engl J Med. 1976 Apr 29;294(18):1013–1013. doi: 10.1056/nejm197604292941823. [DOI] [PubMed] [Google Scholar]
  4. Fendrich G., Abeles R. H. Mechanism of action of butyryl-CoA dehydrogenase: reactions with acetylenic, olefinic, and fluorinated substrate analogues. Biochemistry. 1982 Dec 21;21(26):6685–6695. doi: 10.1021/bi00269a011. [DOI] [PubMed] [Google Scholar]
  5. Freund K., Mizzer J., Dick W., Thorpe C. Inactivation of general acyl-CoA dehydrogenase from pig kidney by 2-alkynoyl coenzyme A derivatives: initial aspects. Biochemistry. 1985 Oct 8;24(21):5996–6002. doi: 10.1021/bi00342a046. [DOI] [PubMed] [Google Scholar]
  6. Gibson K. M., Breuer J., Nyhan W. L. 3-Hydroxy-3-methylglutaryl-coenzyme A lyase deficiency: review of 18 reported patients. Eur J Pediatr. 1988 Dec;148(3):180–186. doi: 10.1007/BF00441397. [DOI] [PubMed] [Google Scholar]
  7. Hruz P. W., Narasimhan C., Miziorko H. M. 3-Hydroxy-3-methylglutaryl coenzyme A lyase: affinity labeling of the Pseudomonas mevalonii enzyme and assignment of cysteine-237 to the active site. Biochemistry. 1992 Jul 28;31(29):6842–6847. doi: 10.1021/bi00144a026. [DOI] [PubMed] [Google Scholar]
  8. Husain S. S., Lowe G. Evidence for histidine in the active sites of ficin and stem-bromelain. Biochem J. 1968 Nov;110(1):53–57. doi: 10.1042/bj1100053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kramer P. R., Miziorko H. M. 3-hydroxy-3-methylglutaryl-CoA lyase: catalysis of acetyl coenzyme A enolization. Biochemistry. 1983 May 10;22(10):2353–2357. doi: 10.1021/bi00279a008. [DOI] [PubMed] [Google Scholar]
  10. Kramer P. R., Miziorko H. M. Purification and characterization of avian liver 3-hydroxy-3-methylglutaryl coenzyme A lyase. J Biol Chem. 1980 Nov 25;255(22):11023–11028. [PubMed] [Google Scholar]
  11. Penefsky H. S. Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase. J Biol Chem. 1977 May 10;252(9):2891–2899. [PubMed] [Google Scholar]
  12. Porter M. A., Potter M. D., Hartman F. C. Affinity labeling of spinach phosphoribulokinase subsequent to S-methylation at Cys16. J Protein Chem. 1990 Aug;9(4):445–451. doi: 10.1007/BF01024620. [DOI] [PubMed] [Google Scholar]
  13. Prasanna P., Holmlund C. E. Identification in Tetrahymena pyriformis of 3-hydroxy-3-methyl glutaryl coenzyme a lyase: its purification and properties. Int J Biochem. 1987;19(4):385–389. doi: 10.1016/0020-711x(87)90013-9. [DOI] [PubMed] [Google Scholar]
  14. Reisler E., Burke M., Himmelfarb S., Harrington W. F. Spatial proximity of the two essential sulfhydryl groups of myosin. Biochemistry. 1974 Sep 10;13(19):3837–3840. doi: 10.1021/bi00716a001. [DOI] [PubMed] [Google Scholar]
  15. Robinson A. M., Williamson D. H. Physiological roles of ketone bodies as substrates and signals in mammalian tissues. Physiol Rev. 1980 Jan;60(1):143–187. doi: 10.1152/physrev.1980.60.1.143. [DOI] [PubMed] [Google Scholar]
  16. Roossien F. F., van Es-Spiekman W., Robillard G. T. Dimeric enzyme IImtl of the E. coli phosphoenolpyruvate-dependent phosphotransferase system. Cross-linking studies with bifunctional sulfhydryl reagents. FEBS Lett. 1986 Feb 17;196(2):284–290. doi: 10.1016/0014-5793(86)80264-2. [DOI] [PubMed] [Google Scholar]
  17. Scher D. S., Rodwell V. W. 3-Hydroxy-3-methylglutaryl coenzyme A lyase from Pseudomonas mevalonii. Biochim Biophys Acta. 1989 Jun 28;1003(3):321–326. doi: 10.1016/0005-2760(89)90239-7. [DOI] [PubMed] [Google Scholar]
  18. Stegink L. D., Coon M. J. Stereospecificity and other properties of highly purified beta-hydroxy-beta-methylglutaryl coenzyme A cleavage enzyme from bovine liver. J Biol Chem. 1968 Oct 25;243(20):5272–5279. [PubMed] [Google Scholar]
  19. Stoops J. K., Wakil S. J. Animal fatty acid synthetase. A novel arrangement of the beta-ketoacyl synthetase sites comprising domains of the two subunits. J Biol Chem. 1981 May 25;256(10):5128–5133. [PubMed] [Google Scholar]
  20. Ziegler D. M. Role of reversible oxidation-reduction of enzyme thiols-disulfides in metabolic regulation. Annu Rev Biochem. 1985;54:305–329. doi: 10.1146/annurev.bi.54.070185.001513. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES