Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1973 Jul;70(7):2077–2081. doi: 10.1073/pnas.70.7.2077

Activation of a Covalent Enzyme-Substrate Bond by Noncovalent Interaction with an Effector

O P Malhotra 1,2,*, Sidney A Bernhard 1,2,†
PMCID: PMC433669  PMID: 4352970

Abstract

The absorption spectrum of an activesite specific chromophoric acyl enzyme, sturgeon β-(2-furyl)-acryloyl-glyceraldehyde-3-phosphate dehydrogenase, is reported. This acyl enzyme undergoes all of the catalyzed reactions characteristic of the intermediate of the physiological acyl enzyme, 3-phospho-D-glyceroyl-glyceraldehyde-3-phosphate dehydrogenease. The rates of reactions of both these acyl enzymes depend strongly on the extent of interaction of the acyl enzyme with the oxidized coenzyme, NAD+, even where the “redox” properties of the coenzyme are not required. Likewise, the spectral properties of chromophoric acyl enzyme are affected by the extent of bound NAD. Under the pseudophysiological conditions reported herein, there is a stoichiometric limitation of two furylacryloyl-acyl groups per enzyme molecule containing four covalently-equivalent subunits. The binding of NAD both to the apoenzyme and to the diacyl enzyme is heterogeneous: at low extents of NAD occupancy, NAD binding is stronger. The binding to acyl enzyme can be quantitatively described by an enzyme model involving a tetramer with 2-fold symmetry, and consequently containing equal numbers of two classes of sites. NAD binding to difurylacryloyl-enzyme occurs virtually discretely, first to the two unmodified (tight-binding) sites, followed by looser binding to the two acyl-sites. NAD occupancy at these latter sites transforms the chromophoric acyl spectrum from that characteristic of a model furylacryloyl-thiol ester in H2O to a highly perturbed furylacryloyl spectrum characteristic of monomeric native “active-thiol” furylacryloyl-enzymes. Likewise the acyl reactivity towards arsenolysis depends on the extent of NAD bound to the loose sites. Elimination of the tight binding of NAD to the difurylacryloyl enzyme tetramer by alkylation of the remaining two free SH groups with iodoacetate has no apparent influence on the NAD-dependent furylacryloyl-spectral perturbation at the “two equivalent acyl sites,” even though it eliminates the apparent “negative cooperativity” in NAD binding.

Keywords: NAD binding, structural asymmetry, acyl glyceraldehyde 3-phosphate dehydrogenase, negative cooperativity

Full text

PDF
2077

Selected References

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

  1. ALLISON W. S., KAPLAN N. O. THE COMPARATIVE ENZYMOLOGY OF TRIOSEPHOSPHATE DEHYDROGENASE. J Biol Chem. 1964 Jul;239:2140–2152. [PubMed] [Google Scholar]
  2. ATKINSON D. E., HATHAWAY J. A., SMITH E. C. KINETICS OF REGULATORY ENZYMES. KINETIC ORDER OF THE YEAST DIPHOSPHOPYRIDINE NUCLEOTIDE ISOCITRATE DEHYDROGENASE REACTION AND A MODEL FOR THE REACTION. J Biol Chem. 1965 Jun;240:2682–2690. [PubMed] [Google Scholar]
  3. Bernhard S. A., Lau S. J., Noller H. Spectrophotometric identification of acyl enzyme intermediates. Biochemistry. 1965 Jun;4(6):1108–1118. doi: 10.1021/bi00882a020. [DOI] [PubMed] [Google Scholar]
  4. Bernhard S. A., Lau S. Spectrophotometric and structural evidence as to the mechanism of portease catalysis at chemical bonding resolution. Cold Spring Harb Symp Quant Biol. 1972;36:75–83. doi: 10.1101/sqb.1972.036.01.013. [DOI] [PubMed] [Google Scholar]
  5. Bernhard S. A., MacQuarrie R. A. Half-site reactivity and the "induced-fit" hypothesis. J Mol Biol. 1973 Feb 15;74(1):73–78. doi: 10.1016/0022-2836(73)90356-2. [DOI] [PubMed] [Google Scholar]
  6. Bloch W., MacQuarrie R. A., Bernhard S. A. The nucleotide and acyl group content of native rabbit muscle glyceraldehyde 3-phosphate dehydrogenase. J Biol Chem. 1971 Feb 10;246(3):780–790. [PubMed] [Google Scholar]
  7. Charney E., Bernhard S. A. Optical properties and the chemical nature of acyl-chymotrypsin linkages. J Am Chem Soc. 1967 May 24;89(11):2726–2733. doi: 10.1021/ja00987a040. [DOI] [PubMed] [Google Scholar]
  8. Conway A., Koshland D. E., Jr Negative cooperativity in enzyme action. The binding of diphosphopyridine nucleotide to glyceraldehyde 3-phosphate dehydrogenase. Biochemistry. 1968 Nov;7(11):4011–4023. doi: 10.1021/bi00851a031. [DOI] [PubMed] [Google Scholar]
  9. FURFINE C. S., VELICK S. F. THE ACYL-ENZYME INTERMEDIATE AND THE KINETIC MECHANISM OF THE GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE REACTION. J Biol Chem. 1965 Feb;240:844–855. [PubMed] [Google Scholar]
  10. Ferdinand W. The isolation and specific activity of rabbit-muscle glyceraldehyde phosphate dehydrogenase. Biochem J. 1964 Sep;92(3):578–585. doi: 10.1042/bj0920578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. HILVERS A. G., WEENEN J. H. Role of DPN in the mechanism of action of glyceraldehydephosphate dehydrogenase. Biochim Biophys Acta. 1962 Apr 9;58:380–383. doi: 10.1016/0006-3002(62)91034-x. [DOI] [PubMed] [Google Scholar]
  12. Hanson K. R. Conformationally distinct subunits in protein oligomers with dihedral symmetry. J Mol Biol. 1968 Nov 28;38(1):133–136. doi: 10.1016/0022-2836(68)90134-4. [DOI] [PubMed] [Google Scholar]
  13. Henderson R. Structure of crystalline alpha-chymotrypsin. IV. The structure of indoleacryloyl-alpha-chyotrypsin and its relevance to the hydrolytic mechanism of the enzyme. J Mol Biol. 1970 Dec 14;54(2):341–354. doi: 10.1016/0022-2836(70)90434-1. [DOI] [PubMed] [Google Scholar]
  14. KRIMSKY I., RACKER E. SEPARATION OF OXIDATIVE FROM PHOSPHORYLATIVE ACTIVITY BY PROTEOLYSIS OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE. Biochemistry. 1963 May-Jun;2:512–518. doi: 10.1021/bi00903a021. [DOI] [PubMed] [Google Scholar]
  15. Koshland D. E., Jr, Némethy G., Filmer D. Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry. 1966 Jan;5(1):365–385. doi: 10.1021/bi00865a047. [DOI] [PubMed] [Google Scholar]
  16. MONOD J., WYMAN J., CHANGEUX J. P. ON THE NATURE OF ALLOSTERIC TRANSITIONS: A PLAUSIBLE MODEL. J Mol Biol. 1965 May;12:88–118. doi: 10.1016/s0022-2836(65)80285-6. [DOI] [PubMed] [Google Scholar]
  17. MacQuarrie R. A., Bernhard S. A. Subunit conformation and catalytic function in rabbit-muscle glyceraldehyde-3-phosphate dehydrogenase. J Mol Biol. 1971 Jan 28;55(2):181–192. doi: 10.1016/0022-2836(71)90190-2. [DOI] [PubMed] [Google Scholar]
  18. Malhotra O. P., Bernhard S. A. Spectrophotometric identification of an active site-specific acyl glyceraldehyde 3-phosphate dehydrogenase. The regulation of its kinetic and equilibrium properties by coenzyme. J Biol Chem. 1968 Mar 25;243(6):1243–1252. [PubMed] [Google Scholar]
  19. RACKER E., KRIMSKY I. The mechanism of oxidation of aldehydes by glyceralde-hyde-3-phosphate dehydrogenase. J Biol Chem. 1952 Oct;198(2):731–743. [PubMed] [Google Scholar]
  20. Trentham D. R. Rate-determining processes and the number of simultaneously active sties of D-glyceraldehyde 3-phosphate dehydrogenase. Biochem J. 1971 Mar;122(1):71–77. doi: 10.1042/bj1220071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Trentham D. R. Reactions of D-glyceraldehyde 3-phosphate dehydrogenase facilitated by oxidized nicotinamide-adenine dinucleotide. Biochem J. 1971 Mar;122(1):59–69. doi: 10.1042/bj1220059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Watson H. C., Duée E., Mercer W. D. Low resolution structure of glyceraldehyde 3-phosphate dehydrogenase. Nat New Biol. 1972 Nov 29;240(100):130–133. doi: 10.1038/newbio240130a0. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES