Dual Mechanisms for Glucose 6-Phosphate Inhibition of Human Brain Hexokinase
1999; Elsevier BV; Volume: 274; Issue: 44 Linguagem: Inglês
10.1074/jbc.274.44.31155
ISSN1083-351X
AutoresXiaofeng Liu, Chang Sup Kim, Feruz T. Kurbanov, Richard B. Honzatko, Herbert J. Fromm,
Tópico(s)Neonatal Health and Biochemistry
ResumoBrain hexokinase (HKI) is inhibited potently by its product glucose 6-phosphate (G6P); however, the mechanism of inhibition is unsettled. Two hypotheses have been proposed to account for product inhibition of HKI. In one, G6P binds to the active site (the C-terminal half of HKI) and competes directly with ATP, whereas in the alternative suggestion the inhibitor binds to an allosteric site (the N-terminal half of HKI), which indirectly displaces ATP from the active site. Single mutations within G6P binding pockets, as defined by crystal structures, at either the N- or C-terminal half of HKI have no significant effect on G6P inhibition. On the other hand, the corresponding mutations eliminate product inhibition in a truncated form of HKI, consisting only of the C-terminal half of the enzyme. Only through combined mutations at the active and allosteric sites, using residues for which single mutations had little effect, was product inhibition eliminated in HKI. Evidently, potent inhibition of HKI by G6P can occur from both active and allosteric binding sites. Furthermore, kinetic data reported here, in conjunction with published equilibrium binding data, are consistent with inhibitory sites of comparable affinity linked by a mechanism of negative cooperativity. Brain hexokinase (HKI) is inhibited potently by its product glucose 6-phosphate (G6P); however, the mechanism of inhibition is unsettled. Two hypotheses have been proposed to account for product inhibition of HKI. In one, G6P binds to the active site (the C-terminal half of HKI) and competes directly with ATP, whereas in the alternative suggestion the inhibitor binds to an allosteric site (the N-terminal half of HKI), which indirectly displaces ATP from the active site. Single mutations within G6P binding pockets, as defined by crystal structures, at either the N- or C-terminal half of HKI have no significant effect on G6P inhibition. On the other hand, the corresponding mutations eliminate product inhibition in a truncated form of HKI, consisting only of the C-terminal half of the enzyme. Only through combined mutations at the active and allosteric sites, using residues for which single mutations had little effect, was product inhibition eliminated in HKI. Evidently, potent inhibition of HKI by G6P can occur from both active and allosteric binding sites. Furthermore, kinetic data reported here, in conjunction with published equilibrium binding data, are consistent with inhibitory sites of comparable affinity linked by a mechanism of negative cooperativity. Mammals harbor four hexokinase (ATP:d-hexose 6-phosphotransferase (2.7.1.1)) isozymes (1Gonzalez C. Ureta T. Sánchez R. Niemeyer H. Biochem. Biophys. Res. Commun. 1964; 16: 347-352Crossref PubMed Scopus (89) Google Scholar, 2Grossbard L. Schimke R.T. J. Biol. Chem. 1966; 241: 3546-3560Abstract Full Text PDF PubMed Google Scholar, 3Katzen H.M. Adv. Enzyme Regul. 1967; 5: 335-356Crossref PubMed Scopus (135) Google Scholar). One of these, brain hexokinase (HKI), 1The abbreviations used are:HKIhexokinase IHKIIhexokinase IImini-HKIC-terminal half of brain hexokinaseG6Pglucose 6-phosphateAnG6P1,5-anhydroglucitol 6-phosphate is putatively the pacemaker of glycolysis in brain tissue and the red blood cell (4Lowry O.H. Passonneau J.V. J Biol. Chem. 1964; 239: 31-42Abstract Full Text PDF PubMed Google Scholar). Two isozymes, HKI and skeletal muscle hexokinase (HKII), are bound to the outer membrane of mitochondria and, in the case of HKI, are juxtaposed to a porin-adenylate translocator complex (5Rose I.A. Warms J.V.B. J. Biol. Chem. 1967; 242: 1635-1645Abstract Full Text PDF PubMed Google Scholar, 6Lindén M. Gellerfors P. Nelson R.D. FEBS Lett. 1982; 141: 189-192Crossref PubMed Scopus (186) Google Scholar, 7Fiek C. Benz R. Roos N. Brdiczka D. Biochim. Biophys. Acta. 1982; 688: 429-440Crossref PubMed Scopus (220) Google Scholar). Only a small fraction of the potential HKI activity is used in brain tissue because of low concentrations of intracellular glucose and potent product inhibition by glucose 6-phosphate (G6P) (8Crane R.K. Boyer P.D. Myrback K. Lardy H.A. The Enzymes. 2nd Ed. 6. Academic Press, New York1962: 47-66Google Scholar, 9Purich D, L. Fromm H.J. J. Biol. Chem. 1971; 246: 3456-3463Abstract Full Text PDF PubMed Google Scholar). Although HKII and HKI are both markedly inhibited by G6P, orthophosphate (Pi) reverses G6P inhibition of only HKI (10Tiedemann H. Born J. Z. Naturforsch. 1959; 146: 477-478Crossref Scopus (12) Google Scholar). In addition, Pi reverses G6P-induced release of mitochondrially bound HKI (5Rose I.A. Warms J.V.B. J. Biol. Chem. 1967; 242: 1635-1645Abstract Full Text PDF PubMed Google Scholar). Exactly how G6P functions as an inhibitor of HKI is unsettled (11Purich D.L. Fromm H.J. Rudolph F.B. Adv. Enzymol. Relat. Areas Mol. Biol. 1973; 39: 249-326PubMed Google Scholar, 12Wilson J.E. Rev. Physiol. Biochem. Pharmacol. 1995; 126: 65-198Crossref PubMed Google Scholar). Although most investigators now believe that G6P competes with ATP at the active site of the enzyme (13Fromm H.J. Zewe V. J. Biol. Chem. 1962; 237: 1661-1667Abstract Full Text PDF PubMed Google Scholar, 14Arora K.K. Filburn C.R. Pederson P.L. J. Biol. Chem. 1993; 268: 18259-18266Abstract Full Text PDF PubMed Google Scholar, 15Magnani M. Bianchi M. Casabianca A. Stocchi V. Danielle A. Altrude F. Ferrone M. Silengo L. Biochem. J. 1992; 285: 193-199Crossref PubMed Scopus (39) Google Scholar, 16Jarori G.K. Iyer S.B. Kasturi S.R. Kendare U.W. Eur. J. Biochem. 1990; 188: 9-14Crossref PubMed Scopus (6) Google Scholar, 17Mehta A. Jarori G.K. Kenkare U.W. J. Biol. Chem. 1988; 263: 15492-15497Abstract Full Text PDF PubMed Google Scholar), others suggest that G6P exerts its effect by binding to an allosteric site topologically distinct from the active site (12Wilson J.E. Rev. Physiol. Biochem. Pharmacol. 1995; 126: 65-198Crossref PubMed Google Scholar, 18Crane R.K. Sols A. J. Biol. Chem. 1954; 210: 597-606Abstract Full Text PDF PubMed Google Scholar, 19Sols A. Kornberg A. Horecker B.L. Cornudella L. Oro J. Reflections on Biochemistry. Pergamon Press, New York1976: 199-206Crossref Google Scholar). On the other hand, there seems to be general agreement regarding the kinetic mechanism of HKI as being rapid-equilibrium Random Bi Bi (20Ning J. Purich D.L. Fromm H.J. J. Biol. Chem. 1969; 244: 3840-3846Abstract Full Text PDF PubMed Google Scholar, 21Bachelard H.S. Clark A.G. Thompson M.F. Biochem. J. 1971; 123: 707-715Crossref PubMed Scopus (60) Google Scholar, 22Gerber G. Preissler H. Heinrich R. Rapoport S.M. Eur. J. Biochem. 1974; 45: 39-52Crossref PubMed Scopus (95) Google Scholar). hexokinase I hexokinase II C-terminal half of brain hexokinase glucose 6-phosphate 1,5-anhydroglucitol 6-phosphate HKI arose putatively from the duplication and fusion of a primordial gene (23Colowick S.P. Boyer P.D. The Enzymes. 3rd Ed. 9. 1973: 1-48Google Scholar). Human HKI has a molecular mass of 100 kDa composed of two structurally similar halves. The two halves (C-terminal and N-terminal) share significant sequence homology (24Nishi S. Seino S. Bell G.I. Bichem. Biophys. Res. Commun. 1988; 157: 937-943Crossref PubMed Scopus (100) Google Scholar). Catalytic activity of the enzyme is associated with the C-terminal half of HKI (14Arora K.K. Filburn C.R. Pederson P.L. J. Biol. Chem. 1993; 268: 18259-18266Abstract Full Text PDF PubMed Google Scholar, 15Magnani M. Bianchi M. Casabianca A. Stocchi V. Danielle A. Altrude F. Ferrone M. Silengo L. Biochem. J. 1992; 285: 193-199Crossref PubMed Scopus (39) Google Scholar, 25White T.K. Wilson J.E. Arch. Biochem. Biophys. 1987; 259: 402-411Crossref PubMed Scopus (53) Google Scholar, 26White T.K. Wilson J.E. Arch. Biochem. Biophys. 1989; 274: 375-393Crossref PubMed Scopus (77) Google Scholar), whereas the N-terminal half has a high affinity site for Piputatively responsible for the relief of G6P inhibition (14Arora K.K. Filburn C.R. Pederson P.L. J. Biol. Chem. 1993; 268: 18259-18266Abstract Full Text PDF PubMed Google Scholar, 15Magnani M. Bianchi M. Casabianca A. Stocchi V. Danielle A. Altrude F. Ferrone M. Silengo L. Biochem. J. 1992; 285: 193-199Crossref PubMed Scopus (39) Google Scholar). Aroraet al. (14Arora K.K. Filburn C.R. Pederson P.L. J. Biol. Chem. 1993; 268: 18259-18266Abstract Full Text PDF PubMed Google Scholar) have suggested that the binding of G6P to this site releases HKI from mitochondria and is not involved in inhibition. Recently published three-dimensional structures of human (27Aleshin A. Zeng C. Bartunik H.D. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1998; 282: 345-357Crossref PubMed Scopus (77) Google Scholar, 28Aleshin A. Zeng C. Bourenkov G.P. Bartunik H.D. Fromm H.J. Honzatko R.B. Structure. 1998; 6: 39-50Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 29Aleshin A.E. Fromm H.J. Honzatko R.B. FEBS Lett. 1998; 434: 42-46Crossref PubMed Scopus (18) Google Scholar) and rat (30Mulichak A.M. Wilson J.E. Padmanabhan K. Garavito R.M. Nat. Struct. Biol. 1998; 5: 555-560Crossref PubMed Scopus (92) Google Scholar) HKI by x-ray crystallography reveals two globular halves held together by a connecting helix and a few hydrogen bonds. Each half is structurally similar to yeast hexokinase. In addition, the crystal structures revealed binding sites for G6P (28Aleshin A. Zeng C. Bourenkov G.P. Bartunik H.D. Fromm H.J. Honzatko R.B. Structure. 1998; 6: 39-50Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 30Mulichak A.M. Wilson J.E. Padmanabhan K. Garavito R.M. Nat. Struct. Biol. 1998; 5: 555-560Crossref PubMed Scopus (92) Google Scholar) and Pi(27Aleshin A. Zeng C. Bartunik H.D. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1998; 282: 345-357Crossref PubMed Scopus (77) Google Scholar). G6P binds to almost identical pockets at the C- and N-terminal halves of HKI, whereas the functional Pi site overlaps the 6-phosphoryl binding locus for G6P at the N-terminal half. Kinetic studies indicate the presence of both high and low affinity binding sites for G6P on HKI (31Fang T.Y. Alechina O. Alechin A.E. Fromm H.J. Honzatko R.B. J. Biol. Chem. 1998; 273: 19548-19553Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Presented here are the kinetic properties of several mutant forms of HKI, in which specific residues (individually and in combination) at G6P pockets, are altered. The results support the following model: (i) G6P binding to high affinity sites at either the N- or C-terminal pocket can independently cause potent inhibition of HKI. (ii) G6P binding to HKI must be strongly anti-cooperative. A full-length cDNA of human brain hexokinase cloned into an expression vector pET-11a (from Novagen) to produce pET-11a-HKI and pET-11d-miniHK was available for use from a previous study (32Zeng C. Fromm H.J. J. Biol. Chem. 1995; 270: 10509-10513Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 33Liu F. Dong Q. Myers A.M. Fromm H.J. Biochem. Biophys. Res. Commun. 1991; 177: 305-311Crossref PubMed Scopus (33) Google Scholar). The transformer site-directed mutagenesis kit is fromCLONTECH. T4 polynucleotide kinase and all the restriction enzymes are from Promega. Bio-gel hydroxyapatite resin is from Bio-Rad. Toyopearl DEAE-650M is from Tosohaas. Oligonucleotide synthesis and DNA sequencing were done at the Iowa State University Nucleic Acid Facility. Escherichia coli strain ZSC13 (DE3), which does not contain endogenous hexokinase, was a gift from the Genetic Stock Center, Yale University. ATP, NADP, 1,5-anhydro-d-sorbitol, deoxyribonuclease (DNase I), leupeptin, phenylmethylsulfonyl fluoride, and ampicillin are from Sigma. Glucose-6-phosphate dehydrogenase came from Roche Molecular Biochemicals. Isopropyl-1-thio-β-d-galactopyranoside is from BioWorld. The hexokinase gene was mutated according to the protocols of the CLONTECH transformer site-directed mutagenesis kit. The mutant plasmid was selected from wild-type plasmids by switching a unique NruI restriction site on the pET-11 vector to another unique XhoI site for the single point mutations. Double mutants were constructed by performing another single mutation in existing single-mutant plasmids. The primers for site-directed mutagenesis are 5′-GATCTTGGAGGAGCAAATTTCCGTG-3′ for Thr536 → Ala, 5′-CTGGATCTTGGTTACTCTTCCTTTCGAATTC-3′ for Gly87 → Tyr, 5′-CTGTGGGAGTGGCAGGGACACTCTAC-3′ for Asp861 → Ala, 5′-CTGATCATCGGCGCTGGCACCAATGC-3′ for Thr232 → Ala, and 5′CTTGGCCCTGGCTCTTGGAGGAACC-3′ for Asp532 → Ala, where the modified codons are bold and underlined. The oligonucleotide primers used for the selection of the mutant plasmid from the wild-type plasmid are: 5′-CAGCCTCGCCTCGAGAACGCCAGCAAG-3′ for the conversion from the NruI site to theXhoI site and 5′-CCTCGCGTCGCGAACGCCAGCAAG-3′ for the conversion from the XhoI site back to the NruI site. Mutations were confirmed by sequencing the entire cDNA insert and coding for HKI. Transformed E. coli strain ZSC13, containing wild-type or mutant pET-11a-HKI, was grown in LB media at 37 °C to an A 600 of 0.6; whereupon the temperature was reduced to 22 °C, and isopropyl-1-thio-β-d-galactopyranoside was added to a final concentration of 0.4 mm. 16–24 h after induction, the cells were harvested and then resuspended in 25 mmKPi (pH 7.5), 2 mm glucose, 1 mmEDTA, 0.4 mm 2-mercaptoethanol, 1 mmphenylmethylsulfonyl fluoride, and 3000 units of DNase I at a temperature of 4 °C. The cells were broken using a French press and centrifuged, after which the supernatant fluid was passed through a DEAE column using a KPi-buffered (pH 7.5), KCl gradient from 0 to 0.5 m. The fractions containing HKI were concentrated and then passed though a hydroxyapatite column using a KPi-buffered (pH 7.5), KCl gradient from 20 to 500 mm. Pooled fractions of HKI were further purified by preparative DEAE-high pressure liquid chromatography, as described elsewhere (31Fang T.Y. Alechina O. Alechin A.E. Fromm H.J. Honzatko R.B. J. Biol. Chem. 1998; 273: 19548-19553Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). AnG6P was prepared as described elsewhere (34Ferrari R.A. Mandelstam P. Crane R.K. Arch. Biochem. Biophys. 1959; 80: 372-377Crossref Scopus (27) Google Scholar). Commercial glucose-6-phosphate dehydrogenase comes as an ammonium sulfate precipitate. Sulfate anion mimics the effect of Pi relief of G6P inhibition (26White T.K. Wilson J.E. Arch. Biochem. Biophys. 1989; 274: 375-393Crossref PubMed Scopus (77) Google Scholar). Thus, to avoid interference from sulfate, glucose-6-phosphate dehydrogenase was dialyzed against the activity assay buffer prior to use in kinetic experiments. HKI activity was determined by the glucose-6-phosphate dehydrogenase-coupled spectrometric assay (13Fromm H.J. Zewe V. J. Biol. Chem. 1962; 237: 1661-1667Abstract Full Text PDF PubMed Google Scholar). Hexokinase concentrations were determined by Bradford assays using bovine serum albumin as a standard (35Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar). Initial rate data were analyzed by using a computer program written in MINITAB with an α-value of 2.0 (36Siano D.B. Zyskind J.W. Fromm H.J. Arch. Biochem. Biophys. 1975; 170: 587-600Crossref PubMed Scopus (58) Google Scholar). In experiments with AnG6P, the kinetic data were fit to a model for nonlinear competitive inhibition with respect to ATP, in which two molecules of inhibitor interact sequentially with HKI (29Aleshin A.E. Fromm H.J. Honzatko R.B. FEBS Lett. 1998; 434: 42-46Crossref PubMed Scopus (18) Google Scholar). This model, which hereafter we will call the stoichiometric model, can be used to evaluate either a system with two independent inhibitor sites or a system with two inhibitor sites coupled by a mechanism of anticooperatively. The equilibrium constants for the dissociation of the first inhibitor molecule from HKI (K i) and the second inhibitor molecule (K ii) take on significantly different meanings in relation to site-specific affinity constants, as discussed below. Circular dichroism spectra were measured from 200 to 260 nm at room temperature by using a Jasco J710 circular dichroism spectrometer. The concentration of HKI used for circular dichroism measurements was 0.2 mg/ml in a buffer containing 2 mm Hepes (pH 7.8), 0.2 mm glucose, and 0.2 mm β-mercaptoethanol. In previous work (31Fang T.Y. Alechina O. Alechin A.E. Fromm H.J. Honzatko R.B. J. Biol. Chem. 1998; 273: 19548-19553Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) we mutated residues at the putative allosteric G6P pocket and found only a modest change (2-fold or less) in the K i for G6P. Subsequently, Sebastian et al. (37Sebastian S.S. Wilson J.E. Malichak A. Garavito R.M. Arch. Biochem. Biophys. 1999; 362: 203-210Crossref PubMed Scopus (15) Google Scholar) mutated the same residues at this site and obtained similar results but concluded that this site was the high affinity binding site for G6P responsible for HKI inhibition. The results from both studies are summarized in Table I. In light of these divergent conclusions, we examined the functional consequences of mutations at the G6P binding site at the C-terminal half of HKI. Our findings and conclusions are the subject of this report.Table IKinetic parameters for mutations in the N-terminal half of hexokinase IEnzymek catK mATPK mGlcK iAnG6PSec−1mmμmμmWild-type HKIaFrom Ref. 31.63.6 ± 3.00.68 ± 0.0453 ± 137 ± 2Asp84 → AlaaFrom Ref. 31.62.2 ± 1.20.79 ± 0.0750 ± 161 ± 2Gly87 → TyraFrom Ref. 31.57.2 ± 5.51.4 ± 0.0138 ± 231 ± 1Ser88 → AlaaFrom Ref. 31.72.3 ± 4.30.59 ± 0.0249 ± 246 ± 2Thr232 → AlaaFrom Ref. 31.70.1 ± 1.80.81 ± 0.0642 ± 284 ± 5Asp84 → AlabFrom Ref. 37.N.P.cEnzyme not purified.0.43 ± 0.0735 ± 233 ± 2Asp84 → GlubFrom Ref. 37.N.P.cEnzyme not purified.0.47 ± 0.0735 ± 618 ± 1Asp84 → LysbFrom Ref. 37.N.P.cEnzyme not purified.0.44 ± 0.0537 ± 328 ± 3a From Ref. 31Fang T.Y. Alechina O. Alechin A.E. Fromm H.J. Honzatko R.B. J. Biol. Chem. 1998; 273: 19548-19553Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar.b From Ref. 37Sebastian S.S. Wilson J.E. Malichak A. Garavito R.M. Arch. Biochem. Biophys. 1999; 362: 203-210Crossref PubMed Scopus (15) Google Scholar.c Enzyme not purified. Open table in a new tab The purity of wild-type and mutant hexokinases was greater than 95% as judged by SDS-polyacrylamide gel electrophoresis (data not shown). Circular dichroism spectra of mutant hexokinases and their cognate, wild-type forms are essentially identical (data not shown) indicating no significant disruption of secondary structure or protein folding because of mutations. The results in Tables Table I, Table II, Table III come from double reciprocal plots of reciprocal initial velocity versus reciprocal substrate concentration (data not shown). The data were subjected to "goodness-of-fit" analysis (36Siano D.B. Zyskind J.W. Fromm H.J. Arch. Biochem. Biophys. 1975; 170: 587-600Crossref PubMed Scopus (58) Google Scholar) using a variety of kinetic models. In all cases the G6P analog, AnG6P, which mimics the properties of G6P in assays of HKI (34Ferrari R.A. Mandelstam P. Crane R.K. Arch. Biochem. Biophys. 1959; 80: 372-377Crossref Scopus (27) Google Scholar), is a competitive inhibitor with respect to ATP and a noncompetitive inhibitor relative to glucose. Kinetic parameters were obtained from the best-fit models, which registered goodness-of-fit values below 5%. Fig.1 illustrates the structure of HKI with G6P bound at the active and allosteric sites. The illustration is based on a 1.9 Å resolution structure of a HKI monomer, which will be presented in detail elsewhere. Asp532 of the C-terminal half interacts with the 2-hydroxyl group of G6P, whereas Asp84, the residue in the N-terminal half corresponding to Asp532, also interacts with the 2-hydroxy group of G6P. Asp861 and Thr680, residues of the C-terminal half, interact with the 1-hydroxyl group and 2-hydroxyl of G6P, respectively. Thr232 interacts with the 6-phosphoryl group of G6P in the N-terminal half of HKI and corresponds structurally to Thr680 of the C-terminal half. Ser88 of the N-terminal half corresponds to Thr536 of the C-terminal half, which hydrogen bonds to the 6-phosphoryl group of G6P. Gly87 of the N-terminal half can accommodate a mutation to a bulky side chain, which should block the binding of Pi or G6P, as noted previously (31Fang T.Y. Alechina O. Alechin A.E. Fromm H.J. Honzatko R.B. J. Biol. Chem. 1998; 273: 19548-19553Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Shown in Tables Table I, Table II, Table III are the results from single mutations of HKI and a form of HKI in which the N-terminal half is absent because of a truncation of the gene that codes for HKI. Hereafter we will refer to this truncated form of HKI as mini-HKI. Single mutations in HKI, either at the allosteric site (Table I) or the G6P binding locus at the active site (Table II) generally cause modest increases (2-fold or less) in the K i for G6P. On the other hand, the same mutations, when made in the putative binding locus of G6P at the active site of mini-HKI, eliminate G6P inhibition (TableIII). These rather surprising and inexplicable results prompted a series of double mutations in HKI, which altered one residue in each of two G6P binding sites (combined N- and C-terminal half mutations). The results of these experiments are in Table III. Evidently, elimination of G6P inhibition comes about only as a consequence of mutations at both G6P binding sites. Evidently, both of the G6P binding sites (allosteric and active site) are functional in HKI, and G6P binding to either causes potent inhibition.Table IIKinetic parameters for mutations in the C-terminal half of hexokinase IEnzymeSpecific activityk catK mATPK mGlcK iAnG6Punits/mg proteinSec−1mmμmμmWild-type HKI61.2 ± 0.9102 ± 20.44 ± 0.0741 ± 218 ± 2Thr536 → Ala5.8 ± 0.059.7 ± 0.081.80 ± 0.0643 ± 125 ± 2Asp861 → Ala1.9 ± 0.13.2 ± 0.20.93 ± 0.1332 ± 229 ± 3Asp532 → LysaFrom Ref. 48.Not available0.0290.57 ± 0.0671 ± 625 ± 9Thr680 → ValaFrom Ref. 48.Not available0.0130.27 ± 0.0433 ± 445 ± 11a From Ref. 48Zeng C. Aleshin A. Hardie J.B. Harrison R.W. Fromm H.J. Biochemistry. 1996; 35: 13157-13164Crossref PubMed Scopus (42) Google Scholar. Open table in a new tab Table IIIG6P binding site mutations in mini-hexokinase and combined mutations in both N- and C-terminal halves of hexokinase IEnzymeSpecific activityk catK mATPK mGlcK iAnG6Punits/mg proteinSec−1mmμmμmMini-HKI, wild-type55 ± 145.6 ± 0.90.50 ± 0.1252 ± 425 ± 2Mini-HKI, Thr536 → Ala13.2 ± 211 ± 10.37 ± 0.0357 ± 3N.D.aNo inhibition by AnG6P for concentrations of at least 500 μm.Mini-HKI, Asp861 → Ala6.4 ± 0.35.3 ± 0.30.85 ± 0.0273 ± 2N.D.aNo inhibition by AnG6P for concentrations of at least 500 μm.Mini-HKI, Asp532→ Ala9.5 ± 0.67.9 ± 0.50.36 ± 0.0838 ± 3N.D.aNo inhibition by AnG6P for concentrations of at least 500 μm.Thr232 → Ala/Thr536 → Ala10.4 ± 0.417.3 ± 0.70.90 ± 0.0361 ± 5N.D.aNo inhibition by AnG6P for concentrations of at least 500 μm.Gly87 → Tyr/Thr536 → Ala3.2 ± 0.15.4 ± 0.21.08 ± 0.06108 ± 8N.D.aNo inhibition by AnG6P for concentrations of at least 500 μm.a No inhibition by AnG6P for concentrations of at least 500 μm. Open table in a new tab In 1951 Weil-Malherbe and Bone (38Weil-Malherbe H. Bone A.D. Biochem. J. 1951; 49: 339-347Crossref PubMed Scopus (35) Google Scholar) reported that G6P is a noncompetitive inhibitor with respect to ATP in the HKI reaction. This finding, along with the high level of G6P specificity when compared with that of mannose 6-phosphate and fructose 6-phosphate, led Crane and Sols (18Crane R.K. Sols A. J. Biol. Chem. 1954; 210: 597-606Abstract Full Text PDF PubMed Google Scholar) to suggest that G6P binds to a site other than the active site, i.e. an allosteric locus. This view has been championed by other investigators (19Sols A. Kornberg A. Horecker B.L. Cornudella L. Oro J. Reflections on Biochemistry. Pergamon Press, New York1976: 199-206Crossref Google Scholar), notably Wilson (12Wilson J.E. Rev. Physiol. Biochem. Pharmacol. 1995; 126: 65-198Crossref PubMed Google Scholar). On the other hand, kinetic studies from our laboratory (13Fromm H.J. Zewe V. J. Biol. Chem. 1962; 237: 1661-1667Abstract Full Text PDF PubMed Google Scholar), as well as many others (2Grossbard L. Schimke R.T. J. Biol. Chem. 1966; 241: 3546-3560Abstract Full Text PDF PubMed Google Scholar, 21Bachelard H.S. Clark A.G. Thompson M.F. Biochem. J. 1971; 123: 707-715Crossref PubMed Scopus (60) Google Scholar, 22Gerber G. Preissler H. Heinrich R. Rapoport S.M. Eur. J. Biochem. 1974; 45: 39-52Crossref PubMed Scopus (95) Google Scholar, 39Kosow D.P. Oski F.A. Warms J.V.B. Rose I.A. Arch. Biochem. Biophys. 1973; 157: 114-124Crossref PubMed Scopus (49) Google Scholar, 40Rijksen G. Staal G.E.J. Biochim. Biophys. Acta. 1977; 485: 75-86Crossref PubMed Scopus (40) Google Scholar, 41Vowels D.T. Easterby J.S. Biochim. Biophys. Acta. 1979; 566: 283-295Crossref PubMed Scopus (21) Google Scholar), showed that G6P is a competitive inhibitor with respect to ATP and a noncompetitive inhibitor with respect to glucose. Based upon these observations, we suggested that G6P competes with ATP at the active site, which is precisely what one would expect of a product inhibitor in a rapid equilibrium Random Bi Bi kinetic mechanism. In support of the above were the findings of Sols (19Sols A. Kornberg A. Horecker B.L. Cornudella L. Oro J. Reflections on Biochemistry. Pergamon Press, New York1976: 199-206Crossref Google Scholar), which were subsequently confirmed by Solheim and Fromm (42Solheim L.P. Fromm H.J. Arch. Biochem. Biophys. 1981; 211: 92-99Crossref PubMed Scopus (18) Google Scholar), that the kinetics of the reverse HKI reaction are normal Michaelin with aK m for G6P nearly equal to the K ifor G6P in the forward reaction. A major breakthrough in HKI research occurred when White and Wilson (25White T.K. Wilson J.E. Arch. Biochem. Biophys. 1987; 259: 402-411Crossref PubMed Scopus (53) Google Scholar) cleaved the enzyme by proteolysis into polypeptides of nearly equal mass. Kinetic studies by these investigators (26White T.K. Wilson J.E. Arch. Biochem. Biophys. 1989; 274: 375-393Crossref PubMed Scopus (77) Google Scholar), and subsequently by others (14Arora K.K. Filburn C.R. Pederson P.L. J. Biol. Chem. 1993; 268: 18259-18266Abstract Full Text PDF PubMed Google Scholar, 15Magnani M. Bianchi M. Casabianca A. Stocchi V. Danielle A. Altrude F. Ferrone M. Silengo L. Biochem. J. 1992; 285: 193-199Crossref PubMed Scopus (39) Google Scholar), demonstrated that the C-terminal half of the enzyme contains the active site, whereas the N-terminal half of HKI is inactive. In addition, except for the reversal of G6P inhibition by Pi, the C-terminal half retained all of the kinetic properties of HKI (14Arora K.K. Filburn C.R. Pederson P.L. J. Biol. Chem. 1993; 268: 18259-18266Abstract Full Text PDF PubMed Google Scholar, 15Magnani M. Bianchi M. Casabianca A. Stocchi V. Danielle A. Altrude F. Ferrone M. Silengo L. Biochem. J. 1992; 285: 193-199Crossref PubMed Scopus (39) Google Scholar, 26White T.K. Wilson J.E. Arch. Biochem. Biophys. 1989; 274: 375-393Crossref PubMed Scopus (77) Google Scholar). Hence, many investigators assigned the site for G6P inhibition to the C-terminal half (active site) and the site for Pi relief of G6P inhibition to the N-terminal half (14Arora K.K. Filburn C.R. Pederson P.L. J. Biol. Chem. 1993; 268: 18259-18266Abstract Full Text PDF PubMed Google Scholar, 15Magnani M. Bianchi M. Casabianca A. Stocchi V. Danielle A. Altrude F. Ferrone M. Silengo L. Biochem. J. 1992; 285: 193-199Crossref PubMed Scopus (39) Google Scholar). Our laboratory had suggested in 1975 that Pibinds at an allosteric site on HKI (43Ellison W.R. Lueck J.D. Fromm H.J. J. Biol. Chem. 1975; 250: 1864-1871Abstract Full Text PDF PubMed Google Scholar). The three-dimensional structures of human HKI yielded an unexpected result in that G6P was bound to both the N- and the C-terminal halves of the enzyme (28Aleshin A. Zeng C. Bourenkov G.P. Bartunik H.D. Fromm H.J. Honzatko R.B. Structure. 1998; 6: 39-50Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 30Mulichak A.M. Wilson J.E. Padmanabhan K. Garavito R.M. Nat. Struct. Biol. 1998; 5: 555-560Crossref PubMed Scopus (92) Google Scholar). Earlier studies indicated the binding of only a single molecule of G6P to HKI (43Ellison W.R. Lueck J.D. Fromm H.J. J. Biol. Chem. 1975; 250: 1864-1871Abstract Full Text PDF PubMed Google Scholar, 44Ellison W.R. Lueck J.D. Fromm H.J. Biochim. Biophys. Acta. 1974; 688: 429-440Google Scholar, 45Chou A.C. Wilson J.E. Arch. Biochem. Biophys. 1974; 165: 628-633Crossref PubMed Scopus (25) Google Scholar). Subsequently, Fang et al. (31Fang T.Y. Alechina O. Alechin A.E. Fromm H.J. Honzatko R.B. J. Biol. Chem. 1998; 273: 19548-19553Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) showed that mutations in the G6P binding site of the N-terminal half of HKI caused only modest increases in theK i for G6P (Table I) and concluded that the N-terminal half could not be the site of potent G6P-inhibition. Shortly thereafter, Sebastian et al. (37Sebastian S.S. Wilson J.E. Malichak A. Garavito R.M. Arch. Biochem. Biophys. 1999; 362: 203-210Crossref PubMed Scopus (15) Google Scholar), using recombinant rat HKI from COS cells, obtained similar results because of mutations of the G6P pocket of the N-terminal half of HKI (Table I); however, they concluded that the N-terminal site was indeed responsible for the potent inhibition of HKI by G6P. The mechanism in Scheme FSI was used in the analysis of kinetic results obtained here and in a previous study (31Fang T.Y. Alechina O. Alechin A.E. Fromm H.J. Honzatko R.B. J. Biol. Chem. 1998; 273: 19548-19553Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The rate equation for Scheme FSI is,1v=1Vm1+KsS1+IKi+I2Ki·KiiEquation 1 where I and S represent G6P and ATP, respectively (glucose is saturating and does not appear in Scheme FSI or in Equation 1), V m is the maximal velocity,K s is the Michaelis constant for ATP, andK i and K ii are inhibition constants for the binding of the first and second molecules of G6P, respectively. Scheme FSI is equally valid for the interpretation of kinetic data for inhibitor binding at independent sites with different affinities or for inhibitor binding to sites with identical affinities coupled by a mechanism of negative cooperativity. However, the relationship of K i and K ii to site affinity constants is model dependent. Most importantly,K i does not have the same meaning for the wild-type and single-mutant enzymes. The results of Tables Table I, Table II, Table III are readily explained from the kinetic equation obtained from Scheme FSII.1v=1Vm1+KsS1+IKn+IKc+I2Kn·KncEquation 2 Scheme FSII differs from Scheme FSI in that it explicitly defines binding sites for G6P at the N- and C-terminal halves of HKI. Site-specific constants for the dissociation of G6P from the N- and C-terminal halves are represented by K n andK c, respectively. These constants measure the dissociation of G6P from either the N-terminal half or the C-terminal half, whichever applies, when the alternative site in not occupied by G6P. K nc represents the dissociation of G6P from the C-terminal site, when the N-terminal site is occupied by another molecule of G6P, and K cn represents the dissociation of G6P from the N-terminal site, when the C-terminal site is occupied. Inhibitor binding to the two sites is random rapid-equilibrium. Hence, if one site is impaired by mutation, inhibition occurs by way of the other site. Direct comparison of the kinetic equations based on Scheme FSI and SchemeFSII results in the following relationship.1Ki=1Kn+1KcEquation 3 For single mutants of HKI considered here, eitherK n or K c approaches infinity. Hence, Equation 3 for single mutants reduces to K i =K n or K i = K c, whichever applies. For the wild-type enzyme, however,K n and K c are related toK i (which is the kinetic constant reported in TablesTable I, Table II, Table III) through Equation 3. For wild-type HKI it is not possible to extract unique values for K n andK c from kinetically determined K ivalues unless an additional assumption is made. If we assume in the wild-type enzyme that K n and K care equal, a reasonable assumption given the K ivalues in Tables I and II, then K n =K c = 2K i. Hence, the approximate 2-fold increase in K i because of the mutation of one of the two G6P binding sites is an expected result and does not suggest the dominance of G6P inhibition derived from the N-terminal site, as some have suggested (37Sebastian S.S. Wilson J.E. Malichak A. Garavito R.M. Arch. Biochem. Biophys. 1999; 362: 203-210Crossref PubMed Scopus (15) Google Scholar). Other than the above subtlety, Scheme FSII is relatively straightforward. The binding of I (G6P) occurs at two sites, one in the C-terminal half and the other in the N-terminal half of HKI. As binding constants obtained from kinetics for either HKI or mini-HKI (TablesTable I, Table II, Table III) are similar, Equation 2 predicts no single mutation will alter the kinetics of inhibition appreciably in the full-length enzyme. On the other hand, a single mutation in mini-HKI or a double mutation in the full-length enzyme should eliminate inhibition, as has been observed (Tables II and III). Furthermore, on the basis of Equations 1and 2, mutations made either in the C- or N-terminal half of HKI should effectively eliminate the (I)2 term. Although kinetic (31Fang T.Y. Alechina O. Alechin A.E. Fromm H.J. Honzatko R.B. J. Biol. Chem. 1998; 273: 19548-19553Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) and structural studies (27Aleshin A. Zeng C. Bartunik H.D. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1998; 282: 345-357Crossref PubMed Scopus (77) Google Scholar, 28Aleshin A. Zeng C. Bourenkov G.P. Bartunik H.D. Fromm H.J. Honzatko R.B. Structure. 1998; 6: 39-50Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 30Mulichak A.M. Wilson J.E. Padmanabhan K. Garavito R.M. Nat. Struct. Biol. 1998; 5: 555-560Crossref PubMed Scopus (92) Google Scholar) strongly suggest that there are two binding sites on HKI for G6P and glucose, direct binding experiments implicate a stoichiometry of 1.0 for these ligands (43Ellison W.R. Lueck J.D. Fromm H.J. J. Biol. Chem. 1975; 250: 1864-1871Abstract Full Text PDF PubMed Google Scholar, 44Ellison W.R. Lueck J.D. Fromm H.J. Biochim. Biophys. Acta. 1974; 688: 429-440Google Scholar, 45Chou A.C. Wilson J.E. Arch. Biochem. Biophys. 1974; 165: 628-633Crossref PubMed Scopus (25) Google Scholar). These seemingly divergent findings can be rationalized by assuming a mechanism of negative cooperativity in ligand binding. The kinetic studies of Fang et al. (31Fang T.Y. Alechina O. Alechin A.E. Fromm H.J. Honzatko R.B. J. Biol. Chem. 1998; 273: 19548-19553Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) revealed both weak and strong binding sites for G6P. Fractional saturation (Ÿ) is related to G6P concentration (I),Y¨=K1(I)+K2(I)+2K3(I)21+K1(I)+K2(I)+K3(I)2Equation 4 where K 1 and K 2represent association constants for ligand binding to free HKI andK 3 is the product of association constants that represents the binding of two molecules of G6P to HKI. If negative cooperativity pertains, then K 3 ≪K 1, K 2,Y¨∼K1(I)+K2(I)1+K1(I)+K2(I)=K(I)1+K(I)Equation 5 where K is the sum of constantsK 1 and K 2. Equation 5represents a binding isotherm with a stoichiometry of unity. Independent binding of G6P to the active and allosteric sites of HKI causes potent inhibition of HKI in vitro. HKI in vivo, however, is bound to the outer mitochondrial membrane. If as Arora et al. (14Arora K.K. Filburn C.R. Pederson P.L. J. Biol. Chem. 1993; 268: 18259-18266Abstract Full Text PDF PubMed Google Scholar) suggest the role of bound G6P at the allosteric site is to release HKI from the mitochondria, then G6P should not be bound to the allosteric site of mitochondrially associated HKI. Under these physiological conditions, if G6P inhibition occurs, it will most likely occur at the active site. This observation is bolstered by the recent work of Ardehali et al. (46Ardehali H. Printz R.L. Whitesell R.R. May J.M. Granner D.K. J. Biol. Chem. 1999; 274: 15986-15989Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), which reports G6P levels above 1 mm in perfused rat hearts, even though hexokinase II, present in that tissue, is inhibitedin vitro by G6P at micromolar levels. G6P binds with high affinity to the isolated N-terminal half of HKII and low affinity to the isolated C-terminal half, but in the full-length enzyme high affinity inhibition dominates. These authors speculate, that HKII in its mitochondrially associated state is inhibited weakly by G6P and that interactions between its N- and C-terminal halves, which are putatively responsible for potent G6P inhibition, are absent. In the case of HKI a fail-safe mechanism exists with respect to G6P inhibition; if one mode of G6P inhibition is lost, then the another mode remains, which assures virtually no diminution in G6P inhibition.
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