Skip to main content
SpringerLink
Log in

Kinetic and proteomic analyses of S-nitrosoglutathione-treated hexokinase A: consequences for cancer energy metabolism

  • Published:
Amino Acids Aims and scope Submit manuscript

Summary.

Mammalian hexokinase (HXK) is found at the outer mitochondrial membrane, exposed to mitochondrial oxygen- and nitrogen-radicals. Given the important role of this enzyme in metabolic pathways and diseases, the effect of S-nitrosoglutathione (GSNO) on HXK A structure and activity was studied. To focus on the catalytic domain, yeast HXK A was used because it has a significant homology to the mammalian domain that contains both the regulatory and catalytic sites. Biologically relevant [GSNO]/[HXK] caused a significant decrease in Vmax with glucose (but not with fructose), along with oxidation of 5 Met and nitration of 4 Tyr. Preincubation of HXK with glucose abrogated the effect of GSNO whereas fructose was ineffective. These results are interpreted by considering the tight binding of glucose to the enzyme as opposed to that of fructose. The segment comprised from amino acids 304 to 306 contained the most modifications. Given that this sequence is highly conserved in HXK from various species, a decline in activity is expected when a high-affinity substrate is presented.

Considering that changes in primary structure are envisioned at high [GSNO]/[HXK] ratios, like those present under normal conditions, it could be hypothesized that the high concentration of hexokinase present in fast growing tumors may serve not only to sustain high glycolysis rates, but also to minimize protein damage that might result in activity decline, compromising energy metabolism.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • CM Anderson RE Stenkamp RC McDonald TA Steitz (1978) ArticleTitleA refined model of the sugar binding site of yeast hexokinase B J Mol Biol 123 207–219 Occurrence Handle355645 Occurrence Handle10.1016/0022-2836(78)90321-2 Occurrence Handle1:CAS:528:DyaE1cXlsF2nurk%3D

    Article  PubMed  CAS  Google Scholar 

  • TL Andreone RL Printz SJ Pilkis MA Magnuson DK Granner (1989) ArticleTitleThe amino acid sequence of rat liver glucokinase deduced from cloned cDNA J Biol Chem 264 363–369 Occurrence Handle2909525 Occurrence Handle1:CAS:528:DyaL1MXltlKktQ%3D%3D

    PubMed  CAS  Google Scholar 

  • KK Arora PL Pedersen (1988) ArticleTitleFunctional significance of mitochondrial bound hexokinase in tumor cell metabolism. Evidence for preferential phosphorylation of glucose by intramitochondrially generated ATP J Biol Chem 263 17422–17428 Occurrence Handle3182854 Occurrence Handle1:CAS:528:DyaL1cXlvVyit78%3D

    PubMed  CAS  Google Scholar 

  • KK Arora M Fanciulli PL Pedersen (1990) ArticleTitleGlucose phosphorylation in tumor cells. Cloning, sequencing, and overexpression in active form of a full-length cDNA encoding a mitochondrial bindable form of hexokinase J Biol Chem 265 6481–6488 Occurrence Handle2318862 Occurrence Handle1:CAS:528:DyaK3cXlsVKgsro%3D

    PubMed  CAS  Google Scholar 

  • KK Arora CR Filburn PL Pedersen (1991) ArticleTitleGlucose phosphorylation. Site-directed mutations which impair the catalytic function of hexokinase J Biol Chem 266 5359–5362 Occurrence Handle2005085 Occurrence Handle1:CAS:528:DyaK3MXktVeqs70%3D

    PubMed  CAS  Google Scholar 

  • KK Arora CR Filburn PL Pedersen (1993) ArticleTitleStructure/function relationships in hexokinase. Site-directed mutational analyses and characterization of overexpressed fragments implicate different functions for the N- and C-terminal halves of the enzyme J Biol Chem 268 18259–18266 Occurrence Handle8349702 Occurrence Handle1:CAS:528:DyaK3sXlvFyms7o%3D

    PubMed  CAS  Google Scholar 

  • SE Barrie EA Saad S Ubatuba P Da Silva Lacaz P Harris (1979) ArticleTitleMyocardial enzyme activities in congestive cardiomyopathy Res Commun Chem Pathol Pharmacol 23 375–381 Occurrence Handle461962 Occurrence Handle1:CAS:528:DyaE1MXhvVCgtbc%3D

    PubMed  CAS  Google Scholar 

  • WS Bennett SuffixJr TA Steitz (1978) ArticleTitleGlucose-induced conformational change in yeast hexokinase Proc Natl Acad Sci USA 75 4848–4852 Occurrence Handle283394 Occurrence Handle10.1073/pnas.75.10.4848 Occurrence Handle1:CAS:528:DyaE1MXjsFGruw%3D%3D

    Article  PubMed  CAS  Google Scholar 

  • WS Bennett SuffixJr TA Steitz (1980) ArticleTitleStructure of a complex between yeast hexokinase A and glucose. II. Detailed comparisons of conformation and active site configuration with the native hexokinase B monomer and dimer J Mol Biol 140 211–230 Occurrence Handle7001032 Occurrence Handle10.1016/0022-2836(80)90103-5 Occurrence Handle1:CAS:528:DyaL3cXls1enu78%3D

    Article  PubMed  CAS  Google Scholar 

  • E Bustamante HP Morris PL Pedersen (1981) ArticleTitleEnergy metabolism of tumor cells. Requirement for a form of hexokinase with a propensity for mitochondrial binding J Biol Chem 256 8699–8704 Occurrence Handle7263678 Occurrence Handle1:CAS:528:DyaL3MXltFejtrk%3D

    PubMed  CAS  Google Scholar 

  • SP Colowick (1973) Hexokinases PD Boyer (Eds) Enzymes EditionNumber3 NumberInSeries9 Academic Press New York 1–48

    Google Scholar 

  • RA Darrow SP Colowick (1962) ArticleTitleHexokinase from baker’s yeast Methods Enzymol 5 226–235 Occurrence Handle10.1016/S0076-6879(62)05209-X Occurrence Handle1:CAS:528:DC%2BD1cXhsFGgurs%3D

    Article  CAS  Google Scholar 

  • SL Elfering TM Sarkela C Giulivi (2002) ArticleTitleBiochemistry of mitochondrial nitric-oxide synthase J Biol Chem 277 38079–38086 Occurrence Handle12154090 Occurrence Handle10.1074/jbc.M205256200 Occurrence Handle1:CAS:528:DC%2BD38Xns1Cgurg%3D

    Article  PubMed  CAS  Google Scholar 

  • K Frohlich K Entian D Mecke (1985) ArticleTitleThe primary structure of the yeast hexokinase PII gene (HXK2) which is responsible for glucose repression Gene (Amst) 36 105–111 Occurrence Handle1:STN:280:DyaL28%2Fls12nsQ%3D%3D

    CAS  Google Scholar 

  • C Giulivi A Boveris E Cadenas (1999) Oxygen radicals in mitochondria: critical evaluation of the methodology available for estimating steady-state concentrations of oxygen radicals DL Gilbert CA Colton (Eds) Reactive oxygen species in biological systems: selected topics Plenum Press New York 77–102

    Google Scholar 

  • C Giulivi (2003) ArticleTitleCharacterization and function of mitochondrial nitric-oxide synthase Free Radical Biol Med 34 397–408 Occurrence Handle10.1016/S0891-5849(02)01298-4 Occurrence Handle1:CAS:528:DC%2BD3sXptFGitg%3D%3D

    Article  CAS  Google Scholar 

  • RE Gots SP Bessman (1974) ArticleTitleThe functional compartmentation of mitochondrial hexokinase Arch Biochem Biophys 163 7–14 Occurrence Handle4854848 Occurrence Handle10.1016/0003-9861(74)90448-2 Occurrence Handle1:CAS:528:DyaE2cXls1Gmt7k%3D

    Article  PubMed  CAS  Google Scholar 

  • LD Griffin BD Gelb DA Wheeler D Davison V Adams ER McCabe (1991) ArticleTitleMammalian hexokinase 1: evolutionary conservation and structure to function analysis Genomics 11 1014–1024 Occurrence Handle1783373 Occurrence Handle10.1016/0888-7543(91)90027-C Occurrence Handle1:CAS:528:DyaK38Xlt1Ohsbc%3D

    Article  PubMed  CAS  Google Scholar 

  • BL Gupta M Nehal NZ Baquer (1997) ArticleTitleEffect of experimental diabetes on the activities of hexokinase, glucose-6-phosphate dehydrogenase and catecholamines in rat erythrocytes of different ages Indian J Exp Biol 35 792–795 Occurrence Handle9418381 Occurrence Handle1:CAS:528:DyaK2sXlvFaqs7k%3D

    PubMed  CAS  Google Scholar 

  • T Hart (1985) ArticleTitleSome observations concerning the S-nitroso and S-phenylsulfonyl derivatives of L-cysteine and glutathione Tetrahedron Lett 26 2013–2016 Occurrence Handle10.1016/S0040-4039(00)98368-0 Occurrence Handle1:CAS:528:DyaL2MXkslWluro%3D

    Article  CAS  Google Scholar 

  • MK Johnson (1960) ArticleTitleThe intracellular distribution of glycolytic and other enzymes in rat-brain homogenates and mitochondrial preparations Biochem J 77 610–618 Occurrence Handle13790316 Occurrence Handle1:CAS:528:DyaF3MXktl2kug%3D%3D

    PubMed  CAS  Google Scholar 

  • HM Katzen RT Schimke (1965) ArticleTitleMultiple forms of hexokinase in the rat: tissue distribution, age dependency, and properties Proc Natl Acad Sci USA 54 1218–1225 Occurrence Handle5219826 Occurrence Handle10.1073/pnas.54.4.1218 Occurrence Handle1:CAS:528:DyaF28XivVOlsg%3D%3D

    Article  PubMed  CAS  Google Scholar 

  • E Kopetzki K Entian D Mecke (1985) ArticleTitleComplete nucleotide sequence of the hexokinase PI gene (HXK1) of Saccharomyces cerevisiae Gene (Amst) 39 95–102 Occurrence Handle1:CAS:528:DyaL28XmtlantA%3D%3D

    CAS  Google Scholar 

  • PR Kuser S Krauchenco OAC Antunes I Polikarpov (2000) ArticleTitleThe high resolution crystal structure of yeast hexokinase PII with the correct primary sequence provides new insights into its mechanism of action J Biol Chem 275 20814–20821 Occurrence Handle10749890 Occurrence Handle10.1074/jbc.M910412199 Occurrence Handle1:CAS:528:DC%2BD3cXkvVyht7Y%3D

    Article  PubMed  CAS  Google Scholar 

  • NR Lazarus Ah Ramel YM Rustum EA Barnard (1966) ArticleTitleYeast hexokinase. I. Preparation of the pure enzyme Biochemistry 5 4003–4016 Occurrence Handle10.1021/bi00876a035 Occurrence Handle1:CAS:528:DyaF2sXhs1Krtg%3D%3D

    Article  CAS  Google Scholar 

  • H Ma LM Bloom SE Dakin CT Walsh D Botstein (1989a) ArticleTitleThe 15 N-terminal amino acids of hexokinase II are not required for in vivo function: analysis of a truncated form of hexokinase II in Saccharomyces cerevisiae Proteins 5 218–223 Occurrence Handle10.1002/prot.340050305 Occurrence Handle1:CAS:528:DyaL1MXkslersLs%3D

    Article  CAS  Google Scholar 

  • H Ma LM Bloom Z Zhu CT Walsh D Botstein (1989b) ArticleTitleIsolation and characterization of mutations in the HXK2 gene of Saccharomyces cerevisiae Mol Cell Biol 9 5630–5642 Occurrence Handle1:CAS:528:DyaK3cXjtFGqtg%3D%3D

    CAS  Google Scholar 

  • M Magnani V Stocchi L Cucchiarini G Novelli S Lodi L Isa G Fornaini (1985) ArticleTitleHereditary nonspherocytic hemolytic anemia due to a new hexokinase variant with reduced stability Blood 66 690–697 Occurrence Handle4027385 Occurrence Handle1:STN:280:DyaL2M3otVymtw%3D%3D

    PubMed  CAS  Google Scholar 

  • RC McDonald TA Steitz DM Engelman (1979) ArticleTitleYeast hexokinase in solution exhibits a large conformational change upon binding glucose or glucose 6-phosphate Biochemistry 18 338–342 Occurrence Handle369601 Occurrence Handle10.1021/bi00569a017 Occurrence Handle1:STN:280:DyaE1M7isVKlsw%3D%3D

    Article  PubMed  CAS  Google Scholar 

  • RJ Middleton (1990) ArticleTitleHexokinases and glucokinases Biochem Soc Trans 18 180–183 Occurrence Handle2199258 Occurrence Handle1:CAS:528:DyaK3cXhvFCitbw%3D

    PubMed  CAS  Google Scholar 

  • Y Mochizuki SH Hori (1976) ArticleTitleHexose 6-phosphate dehydrogenase in starfishes Comp Biochem Physiol B 54 489–494 Occurrence Handle939091 Occurrence Handle10.1016/0305-0491(76)90127-9 Occurrence Handle1:CAS:528:DyaE28XlsFGms7k%3D

    Article  PubMed  CAS  Google Scholar 

  • Y Mochizuki SH Hori (1977) ArticleTitlePurification and properties of hexokinase from the starfish, Asterias amurensis J Biochem (Tokyo) 81 1849–1855 Occurrence Handle1:CAS:528:DyaE2sXkvVShsLY%3D

    CAS  Google Scholar 

  • S Otieno AK Bhargava EA Barnard AH Ramel (1975) ArticleTitleYeast hexokinases. VIII. Essential thiols of yeast hexokinase. Alkylation by a substrate-like reagent Biochemistry 14 2403–2410 Occurrence Handle1095053 Occurrence Handle10.1021/bi00682a021 Occurrence Handle1:CAS:528:DyaE2MXkslCmu74%3D

    Article  PubMed  CAS  Google Scholar 

  • DM Parry PL Pedersen (1983) ArticleTitleIntracellular localization and properties of particulate hexokinase in the Novikoff ascites tumor. Evidence for an outer mitochondrial membrane location J Biol Chem 258 10904–10912 Occurrence Handle6885806 Occurrence Handle1:CAS:528:DyaL3sXltlOls7s%3D

    PubMed  CAS  Google Scholar 

  • AH Ramel YM Rustum JG Jones EA Barnard (1971) ArticleTitleYeast hexokinase. IV. Multiple forms of hexokinase in the yeast cell Biochemistry 10 3499–3508 Occurrence Handle4947783 Occurrence Handle10.1021/bi00795a003 Occurrence Handle1:STN:280:DyaE387msVCqsg%3D%3D

    Article  PubMed  CAS  Google Scholar 

  • IA Rose JV Warms (1967) ArticleTitleMitochondrial hexokinase. Release, rebinding, and location J Biol Chem 242 1635–1645 Occurrence Handle4225734 Occurrence Handle1:CAS:528:DyaF2sXptFyltg%3D%3D

    PubMed  CAS  Google Scholar 

  • YM Rustum AH Ramel EA Barnard (1971) ArticleTitleThe preparation of yeast hexokinases Prep Biochem 1 309–329 Occurrence Handle4950103 Occurrence Handle1:CAS:528:DyaE38Xps1Whtg%3D%3D

    PubMed  CAS  Google Scholar 

  • DA Schwab JE Wilson (1989) ArticleTitleComplete amino acid sequence of rat brain hexokinase, deduced from the cloned cDNA, and proposed structure of a mammalian hexokinase Proc Natl Acad Sci USA 86 2563–2567 Occurrence Handle2704734 Occurrence Handle10.1073/pnas.86.8.2563 Occurrence Handle1:CAS:528:DyaL1MXksFeitLg%3D

    Article  PubMed  CAS  Google Scholar 

  • M Steffen TM Sarkela AA Gybina TW Steele NJ Traaseth D Kuehl C Giulivi (2001) ArticleTitleMetabolism of S-nitrosoglutathione in intact mitochondria Biochem J 356 395–402 Occurrence Handle11368766 Occurrence Handle10.1042/0264-6021:3560395 Occurrence Handle1:CAS:528:DC%2BD3MXks1altb8%3D

    Article  PubMed  CAS  Google Scholar 

  • T Ureta (1982) ArticleTitleThe comparative isozymology of vertebrate hexokinases Comp Biochem Physiol 71B 549–555 Occurrence Handle1:CAS:528:DyaL38Xks1Ort74%3D

    CAS  Google Scholar 

  • WN Valentine FA Oski DE Paglia MA Baughan AS Schneider JL Naiman (1967) ArticleTitleHereditary hemolytic anemia with hexokinase deficiency. Role of hexokinase in erythrocyte aging N Engl J Med 276 1–11 Occurrence Handle6015552 Occurrence Handle1:CAS:528:DyaF2sXmtlGjsg%3D%3D Occurrence Handle10.1056/NEJM196701052760101

    Article  PubMed  CAS  Google Scholar 

  • N Vionnet M Stoffel J Takeda K Yasuda GI Bell H Zouali S Lesage G Velho F Iris P Passa P Froguel D Cohen (1992) ArticleTitleNonsense mutation in the glucokinase gene causes early-onset non-insulin-dependent diabetes mellitus Nature 356 721–722 Occurrence Handle1570017 Occurrence Handle10.1038/356721a0 Occurrence Handle1:CAS:528:DyaK38XisFegurw%3D

    Article  PubMed  CAS  Google Scholar 

  • O Warburg K Posener F Negelein (1924) ArticleTitleMetabolism of carcinoma cells Biochem Z 152 309–344 Occurrence Handle1:CAS:528:DyaB2MXhvVanuw%3D%3D

    CAS  Google Scholar 

  • TK White JE Wilson (1987) ArticleTitleRat brain hexokinase: location of the allosteric regulatory site in a structural domain at the N-terminus of the enzyme Arch Biochem Biophys 269 402–411 Occurrence Handle10.1016/0003-9861(87)90506-6

    Article  Google Scholar 

  • TK White JE Wilson (1989) ArticleTitleIsolation and characterization of the discrete N- and C-terminal halves of rat brain hexokinase: retention of full catalytic activity in the isolated C-terminal half Arch Biochem Biophys 274 375–393 Occurrence Handle2802617 Occurrence Handle10.1016/0003-9861(89)90451-7 Occurrence Handle1:CAS:528:DyaL1MXlvFShtbg%3D

    Article  PubMed  CAS  Google Scholar 

  • JE Wilson (2003) ArticleTitleIsozymes of mammalian hexokinase: structure, subcellular localization and metabolic function J Exp Biol 206 2049–2057 Occurrence Handle12756287 Occurrence Handle10.1242/jeb.00241 Occurrence Handle1:CAS:528:DC%2BD3sXlslaltbw%3D

    Article  PubMed  CAS  Google Scholar 

  • FC Womack MK Welch J Nielsen SP Colowick (1973) ArticleTitlePurification and serological comparison of the yeast hexokinases P-I and P-II Arch Biochem Biophys 158 451–457 Occurrence Handle4592979 Occurrence Handle10.1016/0003-9861(73)90536-5 Occurrence Handle1:CAS:528:DyaE2cXisVWntA%3D%3D

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Molecular Biosciences, University of California, Davis, CA, U.S.A.

    S. Miller, C. Ross-Inta & C. Giulivi

Authors
  1. S. Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. C. Ross-Inta
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. C. Giulivi
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Miller, S., Ross-Inta, C. & Giulivi, C. Kinetic and proteomic analyses of S-nitrosoglutathione-treated hexokinase A: consequences for cancer energy metabolism. Amino Acids 32, 593–602 (2007). https://doi.org/10.1007/s00726-006-0424-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00726-006-0424-9

Search

Navigation