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. 1992 Jul;1(7):892–901. doi: 10.1002/pro.5560010707

Charge balance in the alpha-hydroxyacid dehydrogenase vacuole: an acid test.

A Cortes 1, D C Emery 1, D J Halsall 1, R M Jackson 1, A R Clarke 1, J J Holbrook 1
PMCID: PMC2142153  PMID: 1304374

Abstract

The proposal that the active site vacuole of NAD(+)-S-lactate dehydrogenase is unable to accommodate any imbalance in electrostatic charge was tested by genetically manipulating the cDNA coding for human muscle lactate dehydrogenase to make a protein with an aspartic acid introduced at position 140 instead of the wild-type asparagine. The Asn 140-Asp mutant enzyme has the same kcat as the wild type (Asn 140) at low pH (4.5), and at higher pH the Km for pyruvate increases 10-fold for each unit increase in pH up to pH 9. We conclude that the anion of Asp 140 is completely inactive and that it binds pyruvate with a Km that is over 1,000 times that of the Km of the neutral, protonated aspartic-140. Experimental results and molecular modeling studies indicate the pKa of the active site histidine-195 in the enzyme-NADH complex is raised to greater than 10 by the presence of the anion at position 140. Energy minimization and molecular dynamics studies over 36 ps suggest that the anion at position 140 promotes the opening of and the entry of mobile solvent beneath the polypeptide loop (98-110), which normally seals off the internal active site vacuole from external bulk solvent.

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Selected References

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  1. Abad-Zapatero C., Griffith J. P., Sussman J. L., Rossmann M. G. Refined crystal structure of dogfish M4 apo-lactate dehydrogenase. J Mol Biol. 1987 Dec 5;198(3):445–467. doi: 10.1016/0022-2836(87)90293-2. [DOI] [PubMed] [Google Scholar]
  2. Barstow D. A., Black G. W., Sharman A. F., Scawen M. D., Atkinson T., Li S. S., Chia W. N., Clarke A. R., Holbrook J. J. Expression of the copy DNA for human A4 and B4 L-lactate dehydrogenases in Escherichia coli. Biochim Biophys Acta. 1990 Sep 10;1087(1):73–79. doi: 10.1016/0167-4781(90)90123-j. [DOI] [PubMed] [Google Scholar]
  3. CLELAND W. W. The kinetics of enzyme-catalyzed reactions with two or more substrates or products. II. Inhibition: nomenclature and theory. Biochim Biophys Acta. 1963 Feb 12;67:173–187. doi: 10.1016/0006-3002(63)91815-8. [DOI] [PubMed] [Google Scholar]
  4. Clarke A. R., Colebrook S., Cortes A., Emery D. C., Halsall D. J., Hart K. W., Jackson R. M., Wilks H. M., Holbrook J. J. Towards the construction of a universal NAD(P)(+)-dependent dehydrogenase: comparative and evolutionary considerations. Biochem Soc Trans. 1991 Aug;19(3):576–581. doi: 10.1042/bst0190576. [DOI] [PubMed] [Google Scholar]
  5. Clarke A. R., Smith C. J., Hart K. W., Wilks H. M., Chia W. N., Lee T. V., Birktoft J. J., Banaszak L. J., Barstow D. A., Atkinson T. Rational construction of a 2-hydroxyacid dehydrogenase with new substrate specificity. Biochem Biophys Res Commun. 1987 Oct 14;148(1):15–23. doi: 10.1016/0006-291x(87)91070-9. [DOI] [PubMed] [Google Scholar]
  6. Clarke A. R., Wigley D. B., Chia W. N., Barstow D., Atkinson T., Holbrook J. J. Site-directed mutagenesis reveals role of mobile arginine residue in lactate dehydrogenase catalysis. Nature. 1986 Dec 18;324(6098):699–702. doi: 10.1038/324699a0. [DOI] [PubMed] [Google Scholar]
  7. Dauber-Osguthorpe P., Roberts V. A., Osguthorpe D. J., Wolff J., Genest M., Hagler A. T. Structure and energetics of ligand binding to proteins: Escherichia coli dihydrofolate reductase-trimethoprim, a drug-receptor system. Proteins. 1988;4(1):31–47. doi: 10.1002/prot.340040106. [DOI] [PubMed] [Google Scholar]
  8. Dunn C. R., Wilks H. M., Halsall D. J., Atkinson T., Clarke A. R., Muirhead H., Holbrook J. J. Design and synthesis of new enzymes based on the lactate dehydrogenase framework. Philos Trans R Soc Lond B Biol Sci. 1991 May 29;332(1263):177–184. doi: 10.1098/rstb.1991.0047. [DOI] [PubMed] [Google Scholar]
  9. FAWCETT C. P., KAPLAN N. O. Preparation and properties of some nicotinamide adenine dinucleotide analogues with pentose and purine modifications. J Biol Chem. 1962 May;237:1709–1715. [PubMed] [Google Scholar]
  10. Feeney R., Clarke A. R., Holbrook J. J. A single amino acid substitution in lactate dehydrogenase improves the catalytic efficiency with an alternative coenzyme. Biochem Biophys Res Commun. 1990 Jan 30;166(2):667–672. doi: 10.1016/0006-291x(90)90861-g. [DOI] [PubMed] [Google Scholar]
  11. Gilson M. K., Honig B. H. Energetics of charge-charge interactions in proteins. Proteins. 1988;3(1):32–52. doi: 10.1002/prot.340030104. [DOI] [PubMed] [Google Scholar]
  12. Hart K. W., Clarke A. R., Wigley D. B., Waldman A. D., Chia W. N., Barstow D. A., Atkinson T., Jones J. B., Holbrook J. J. A strong carboxylate-arginine interaction is important in substrate orientation and recognition in lactate dehydrogenase. Biochim Biophys Acta. 1987 Aug 21;914(3):294–298. doi: 10.1016/0167-4838(87)90289-5. [DOI] [PubMed] [Google Scholar]
  13. Higuchi R., Krummel B., Saiki R. K. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 1988 Aug 11;16(15):7351–7367. doi: 10.1093/nar/16.15.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  15. Holbrook J. J. Direct measurement of proton binding to the active ternary complex of pig heart lactate dehydrogenase. Biochem J. 1973 Aug;133(4):847–849. doi: 10.1042/bj1330847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Holbrook J. J., Stinson R. A. The use of ternary complexes to study ionizations and isomerizations during catalysis by lactate dehydrogenase. Biochem J. 1973 Apr;131(4):739–748. doi: 10.1042/bj1310739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hurley J. H., Dean A. M., Sohl J. L., Koshland D. E., Jr, Stroud R. M. Regulation of an enzyme by phosphorylation at the active site. Science. 1990 Aug 31;249(4972):1012–1016. doi: 10.1126/science.2204109. [DOI] [PubMed] [Google Scholar]
  18. Klapper I., Hagstrom R., Fine R., Sharp K., Honig B. Focusing of electric fields in the active site of Cu-Zn superoxide dismutase: effects of ionic strength and amino-acid modification. Proteins. 1986 Sep;1(1):47–59. doi: 10.1002/prot.340010109. [DOI] [PubMed] [Google Scholar]
  19. Lodola A., Parker D. M., Jeck R., Holbrook J. J. Malate dehydrogenase of the cytosol. Ionizations of the enzyme-reduced-coenzyme complex and a comparison with lactate dehydrogenase. Biochem J. 1978 Aug 1;173(2):597–605. doi: 10.1042/bj1730597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Parker D. M., Jeckel D., Holbrook J. J. Slow structural changes shown by the 3-nitrotyrosine-237 residue in pig heart [Tyr(3NO2)237] lactate dehydrogenase. Biochem J. 1982 Mar 1;201(3):465–471. doi: 10.1042/bj2010465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Parker D. M., Lodola A., Holbrook J. J. Use of the sulphite adduct of nicotinamide-adenine dinucleotide to study ionizations and the kinetics of lactate dehydrogenase and malate dehydrogenase. Biochem J. 1978 Sep 1;173(3):959–967. doi: 10.1042/bj1730959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sharp K. A., Honig B. Electrostatic interactions in macromolecules: theory and applications. Annu Rev Biophys Biophys Chem. 1990;19:301–332. doi: 10.1146/annurev.bb.19.060190.001505. [DOI] [PubMed] [Google Scholar]
  23. Smith C. J., Clarke A. R., Chia W. N., Irons L. I., Atkinson T., Holbrook J. J. Detection and characterization of intermediates in the folding of large proteins by the use of genetically inserted tryptophan probes. Biochemistry. 1991 Jan 29;30(4):1028–1036. doi: 10.1021/bi00218a021. [DOI] [PubMed] [Google Scholar]
  24. Waldman A. D., Hart K. W., Clarke A. R., Wigley D. B., Barstow D. A., Atkinson T., Chia W. N., Holbrook J. J. The use of genetically engineered tryptophan to identify the movement of a domain of B. stearothermophilus lactate dehydrogenase with the process which limits the steady-state turnover of the enzyme. Biochem Biophys Res Commun. 1988 Jan 29;150(2):752–759. doi: 10.1016/0006-291x(88)90455-x. [DOI] [PubMed] [Google Scholar]
  25. Warshel A., Russell S. T. Calculations of electrostatic interactions in biological systems and in solutions. Q Rev Biophys. 1984 Aug;17(3):283–422. doi: 10.1017/s0033583500005333. [DOI] [PubMed] [Google Scholar]
  26. Wigley D. B., Gamblin S. J., Turkenburg J. P., Dodson E. J., Piontek K., Muirhead H., Holbrook J. J. Structure of a ternary complex of an allosteric lactate dehydrogenase from Bacillus stearothermophilus at 2.5 A resolution. J Mol Biol. 1992 Jan 5;223(1):317–335. doi: 10.1016/0022-2836(92)90733-z. [DOI] [PubMed] [Google Scholar]
  27. Wilks H. M., Halsall D. J., Atkinson T., Chia W. N., Clarke A. R., Holbrook J. J. Designs for a broad substrate specificity keto acid dehydrogenase. Biochemistry. 1990 Sep 18;29(37):8587–8591. doi: 10.1021/bi00489a013. [DOI] [PubMed] [Google Scholar]
  28. Wilks H. M., Hart K. W., Feeney R., Dunn C. R., Muirhead H., Chia W. N., Barstow D. A., Atkinson T., Clarke A. R., Holbrook J. J. A specific, highly active malate dehydrogenase by redesign of a lactate dehydrogenase framework. Science. 1988 Dec 16;242(4885):1541–1544. doi: 10.1126/science.3201242. [DOI] [PubMed] [Google Scholar]

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