Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1995 Oct;4(10):2156–2167. doi: 10.1002/pro.5560041022

The role of glutamate 87 in the kinetic mechanism of Thermus thermophilus isopropylmalate dehydrogenase.

A M Dean 1, L Dvorak 1
PMCID: PMC2142978  PMID: 8535253

Abstract

The kinetic mechanism of the oxidative decarboxylation of 2R,3S-isopropylmalate by the NAD-dependent isopropylmalate dehydrogenase of Thermus thermophilus was investigated. Initial rate results typical of random or steady-state ordered sequential mechanisms are obtained for both the wild-type and two mutant enzymes (E87G and E87Q) regardless of whether natural or alternative substrates (2R-malate, 2R,3S-tartrate and/or NADP) are utilized. Initial rate data fail to converge on a rapid equilibrium-ordered pattern despite marked reductions in specificity (kcat/Km) caused by the mutations and alternative substrates. Although the inhibition studies alone might suggest an ordered kinetic mechanism with cofactor binding first, a detailed analysis reveals that the expected noncompetitive patterns appear uncompetitive because the dissociation constants from the ternary complexes are far smaller than those from the binary complexes. Equilibrium fluorescence studies both confirm the random binding of substrates and the kinetic estimates of the dissociation constants of the substrates from the binary complexes. The latter are not distributed markedly by the mutations at site 87. Mutations at site 87 do not affect the dissociation constants from the binary complexes, but do greatly increase the Michaelis constants, indicating that E87 helps stabilize the Michaelis complex of the wild-type enzyme. The available structural data, the patterns of the kinetics results, and the structure of a pseudo-Michaelis complex of the homologous isocitrate dehydrogenase of Escherichia coli suggest that E87 interacts with the nicotinamide ring.

Full Text

The Full Text of this article is available as a PDF (1.8 MB).

Selected References

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

  1. CLELAND W. W. The kinetics of enzyme-catalyzed reactions with two or more substrates or products. III. Prediction of initial velocity and inhibition patterns by inspection. Biochim Biophys Acta. 1963 Feb 12;67:188–196. doi: 10.1016/0006-3002(63)91816-x. [DOI] [PubMed] [Google Scholar]
  2. Dalziel K., Londesborough J. C. The mechanisms of reductive carboxylation reactions. Carbon dioxide or bicarbonate as substrate of nicotinamide-adenine dinucleotide phosphate-linked isocitrate dehydrogenase and malic enzyme. Biochem J. 1968 Nov;110(2):223–230. doi: 10.1042/bj1100223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dean A. M., Koshland D. E., Jr Electrostatic and steric contributions to regulation at the active site of isocitrate dehydrogenase. Science. 1990 Aug 31;249(4972):1044–1046. doi: 10.1126/science.2204110. [DOI] [PubMed] [Google Scholar]
  4. Dean A. M., Koshland D. E., Jr Kinetic mechanism of Escherichia coli isocitrate dehydrogenase. Biochemistry. 1993 Sep 14;32(36):9302–9309. doi: 10.1021/bi00087a007. [DOI] [PubMed] [Google Scholar]
  5. Dean A. M., Lee M. H., Koshland D. E., Jr Phosphorylation inactivates Escherichia coli isocitrate dehydrogenase by preventing isocitrate binding. J Biol Chem. 1989 Dec 5;264(34):20482–20486. [PubMed] [Google Scholar]
  6. Grissom C. B., Cleland W. W. Isotope effect studies of the chemical mechanism of pig heart NADP isocitrate dehydrogenase. Biochemistry. 1988 Apr 19;27(8):2934–2943. doi: 10.1021/bi00408a040. [DOI] [PubMed] [Google Scholar]
  7. Hurley J. H., Dean A. M., Koshland D. E., Jr, Stroud R. M. Catalytic mechanism of NADP(+)-dependent isocitrate dehydrogenase: implications from the structures of magnesium-isocitrate and NADP+ complexes. Biochemistry. 1991 Sep 3;30(35):8671–8678. doi: 10.1021/bi00099a026. [DOI] [PubMed] [Google Scholar]
  8. Hurley J. H., Dean A. M. Structure of 3-isopropylmalate dehydrogenase in complex with NAD+: ligand-induced loop closing and mechanism for cofactor specificity. Structure. 1994 Nov 15;2(11):1007–1016. doi: 10.1016/s0969-2126(94)00104-9. [DOI] [PubMed] [Google Scholar]
  9. Hurley J. H., Thorsness P. E., Ramalingam V., Helmers N. H., Koshland D. E., Jr, Stroud R. M. Structure of a bacterial enzyme regulated by phosphorylation, isocitrate dehydrogenase. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8635–8639. doi: 10.1073/pnas.86.22.8635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Imada K., Sato M., Tanaka N., Katsube Y., Matsuura Y., Oshima T. Three-dimensional structure of a highly thermostable enzyme, 3-isopropylmalate dehydrogenase of Thermus thermophilus at 2.2 A resolution. J Mol Biol. 1991 Dec 5;222(3):725–738. doi: 10.1016/0022-2836(91)90508-4. [DOI] [PubMed] [Google Scholar]
  11. Kagawa Y., Nojima H., Nukiwa N., Ishizuka M., Nakajima T., Yasuhara T., Tanaka T., Oshima T. High guanine plus cytosine content in the third letter of codons of an extreme thermophile. DNA sequence of the isopropylmalate dehydrogenase of Thermus thermophilus. J Biol Chem. 1984 Mar 10;259(5):2956–2960. [PubMed] [Google Scholar]
  12. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. LIENHARD G. E., ROSE I. A. THE STEREOCHEMISTRY OF DECARBOXYLATION OF ISOCITRATE BY ISOCITRIC ACID DEHYDROGENASE. Biochemistry. 1964 Feb;3:185–190. doi: 10.1021/bi00890a008. [DOI] [PubMed] [Google Scholar]
  14. Miyazaki K., Kakinuma K., Terasawa H., Oshima T. Kinetic analysis on the substrate specificity of 3-isopropylmalate dehydrogenase. FEBS Lett. 1993 Oct 11;332(1-2):35–36. doi: 10.1016/0014-5793(93)80477-c. [DOI] [PubMed] [Google Scholar]
  15. Northrop D. B., Cleland W. W. The kinetics of pig heart triphosphopyridine nucleotide-isocitrate dehydrogenase. II. Dead-end and multiple inhibition studies. J Biol Chem. 1974 May 10;249(9):2928–2931. [PubMed] [Google Scholar]
  16. SIEBERT G., CARSIOTIS M., PLAUT G. W. [The enzymatic properties of isocitric dehydrogenase]. J Biol Chem. 1957 Jun;226(2):977–991. [PubMed] [Google Scholar]
  17. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Tipton P. A., Beecher B. S. Tartrate dehydrogenase, a new member of the family of metal-dependent decarboxylating R-hydroxyacid dehydrogenases. Arch Biochem Biophys. 1994 Aug 15;313(1):15–21. doi: 10.1006/abbi.1994.1352. [DOI] [PubMed] [Google Scholar]
  19. Yamada T., Akutsu N., Miyazaki K., Kakinuma K., Yoshida M., Oshima T. Purification, catalytic properties, and thermal stability of threo-Ds-3-isopropylmalate dehydrogenase coded by leuB gene from an extreme thermophile, Thermus thermophilus strain HB8. J Biochem. 1990 Sep;108(3):449–456. doi: 10.1093/oxfordjournals.jbchem.a123220. [DOI] [PubMed] [Google Scholar]

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

RESOURCES