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
. 1992 Mar;1(3):329–334. doi: 10.1002/pro.5560010304

Lysine-21 of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase participates in substrate binding through charge-charge interaction.

W T Lee 1, H R Levy 1
PMCID: PMC2142207  PMID: 1304341

Abstract

Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PD) was isolated in high yield and purified to homogeneity from a newly constructed strain of Escherichia coli which lacks its own glucose 6-phosphate dehydrogenase gene. Lys-21 is one of two lysyl residues in the enzyme previously modified by the affinity labels pyridoxal 5'-phosphate and pyridoxal 5'-diphosphate-5'-adenosine, which are competitive inhibitors of the enzyme with respect to glucose 6-phosphate (LaDine, J.R., Carlow, D., Lee, W.T., Cross, R.L., Flynn, T.G., & Levy, H.R., 1991, J. Biol. Chem. 266, 5558-5562). K21R and K21Q mutants of the enzyme were purified to homogeneity and characterized kinetically to determine the function of Lys-21. Both mutant enzymes showed increased Km-values for glucose 6-phosphate compared to wild-type enzyme: 1.4-fold (NAD-linked reaction) and 2.1-fold (NADP-linked reaction) for the K21R enzyme, and 36-fold (NAD-linked reaction) and 53-fold (NADP-linked reaction) for the K21Q enzyme. The Km for NADP+ was unchanged in both mutant enzymes. The Km for NAD+ was increased 1.5- and 3.2-fold, compared to the wild-type enzyme, in the K21R and K21Q enzymes, respectively. For the K21R enzyme the kcat for the NAD- and NADP-linked reactions was unchanged. The kcat for the K21Q enzyme was increased in the NAD-linked reaction by 26% and decreased by 30% in the NADP-linked reaction from the values for the wild-type enzyme. The data are consistent with Lys-21 participating in the binding of the phosphate group of the substrate to the enzyme via charge-charge interaction.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

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

  1. Adams M. J., Levy H. R., Moffat K. Crystallization and preliminary x-ray data for glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. J Biol Chem. 1983 May 10;258(9):5867–5868. [PubMed] [Google Scholar]
  2. Barnell W. O., Yi K. C., Conway T. Sequence and genetic organization of a Zymomonas mobilis gene cluster that encodes several enzymes of glucose metabolism. J Bacteriol. 1990 Dec;172(12):7227–7240. doi: 10.1128/jb.172.12.7227-7240.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cleland W. W. Statistical analysis of enzyme kinetic data. Methods Enzymol. 1979;63:103–138. doi: 10.1016/0076-6879(79)63008-2. [DOI] [PubMed] [Google Scholar]
  4. Fersht A. R. Conformational equilibria in -and -chymotrypsin. The energetics and importance of the salt bridge. J Mol Biol. 1972 Mar 14;64(2):497–509. doi: 10.1016/0022-2836(72)90513-x. [DOI] [PubMed] [Google Scholar]
  5. Fouts D., Ganguly R., Gutierrez A. G., Lucchesi J. C., Manning J. E. Nucleotide sequence of the Drosophila glucose-6-phosphate dehydrogenase gene and comparison with the homologous human gene. Gene. 1988 Mar 31;63(2):261–275. doi: 10.1016/0378-1119(88)90530-6. [DOI] [PubMed] [Google Scholar]
  6. Haghighi B., Levy H. R. Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Conformational transitions induced by nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and glucose 6-phosphate monitored by fluorescent probes. Biochemistry. 1982 Dec 7;21(25):6421–6428. doi: 10.1021/bi00268a016. [DOI] [PubMed] [Google Scholar]
  7. Hanukoglu I., Gutfinger T. cDNA sequence of adrenodoxin reductase. Identification of NADP-binding sites in oxidoreductases. Eur J Biochem. 1989 Mar 15;180(2):479–484. doi: 10.1111/j.1432-1033.1989.tb14671.x. [DOI] [PubMed] [Google Scholar]
  8. Ho Y. S., Howard A. J., Crapo J. D. Cloning and sequence of a cDNA encoding rat glucose-6-phosphate dehydrogenase. Nucleic Acids Res. 1988 Aug 11;16(15):7746–7746. doi: 10.1093/nar/16.15.7746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. LaDine J. R., Carlow D., Lee W. T., Cross R. L., Flynn T. G., Levy H. R. Interaction of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase with pyridoxal 5'-diphospho-5'-adenosine. Affinity labeling of Lys-21 and Lys-343. J Biol Chem. 1991 Mar 25;266(9):5558–5562. [PubMed] [Google Scholar]
  10. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  11. Levy H. R., Christoff M., Ingulli J., Ho E. M. Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: revised kinetic mechanism and kinetics of ATP inhibition. Arch Biochem Biophys. 1983 Apr 15;222(2):473–488. doi: 10.1016/0003-9861(83)90546-5. [DOI] [PubMed] [Google Scholar]
  12. Levy H. R., Daouk G. H. Simultaneous analysis of NAD- and NADP-linked activities of dual nucleotide-specific dehydrogenases. Application to Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase. J Biol Chem. 1979 Jun 10;254(11):4843–4847. [PubMed] [Google Scholar]
  13. Merril C. R. Gel-staining techniques. Methods Enzymol. 1990;182:477–488. doi: 10.1016/0076-6879(90)82038-4. [DOI] [PubMed] [Google Scholar]
  14. Nogae I., Johnston M. Isolation and characterization of the ZWF1 gene of Saccharomyces cerevisiae, encoding glucose-6-phosphate dehydrogenase. Gene. 1990 Dec 15;96(2):161–169. doi: 10.1016/0378-1119(90)90248-p. [DOI] [PubMed] [Google Scholar]
  15. Olive C., Geroch M. E., Levy H. R. Glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides. Kinetic studies. J Biol Chem. 1971 Apr 10;246(7):2047–2057. [PubMed] [Google Scholar]
  16. Persico M. G., Viglietto G., Martini G., Toniolo D., Paonessa G., Moscatelli C., Dono R., Vulliamy T., Luzzatto L., D'Urso M. Isolation of human glucose-6-phosphate dehydrogenase (G6PD) cDNA clones: primary structure of the protein and unusual 5' non-coding region. Nucleic Acids Res. 1986 Mar 25;14(6):2511–2522. doi: 10.1093/nar/14.6.2511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Poteete A. R., Sun D. P., Nicholson H., Matthews B. W. Second-site revertants of an inactive T4 lysozyme mutant restore activity by restructuring the active site cleft. Biochemistry. 1991 Feb 5;30(5):1425–1432. doi: 10.1021/bi00219a037. [DOI] [PubMed] [Google Scholar]
  18. Rowley D. L., Wolf R. E., Jr Molecular characterization of the Escherichia coli K-12 zwf gene encoding glucose 6-phosphate dehydrogenase. J Bacteriol. 1991 Feb;173(3):968–977. doi: 10.1128/jb.173.3.968-977.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Wilkinson A. J., Fersht A. R., Blow D. M., Winter G. Site-directed mutagenesis as a probe of enzyme structure and catalysis: tyrosyl-tRNA synthetase cysteine-35 to glycine-35 mutation. Biochemistry. 1983 Jul 19;22(15):3581–3586. doi: 10.1021/bi00284a007. [DOI] [PubMed] [Google Scholar]

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

RESOURCES