The Roles of Glycine Residues in the ATP Binding Site of Human Brain Hexokinase
1998; Elsevier BV; Volume: 273; Issue: 2 Linguagem: Inglês
10.1074/jbc.273.2.700
ISSN1083-351X
AutoresChenbo Zeng, Alexander E. Aleshin, Guanjun Chen, Richard B. Honzatko, Herbert J. Fromm,
Tópico(s)Amino Acid Enzymes and Metabolism
ResumoMutants of hexokinase I (Arg539→ Lys, Thr661 → Ala, Thr661 → Val, Gly534 → Ala, Gly679 → Ala, and Gly862 → Ala), located putatively in the vicinity of the ATP binding pocket, were constructed, purified to homogeneity, and studied by circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and initial velocity kinetics. The wild-type and mutant enzymes have similar secondary structures on the basis of CD spectroscopy. The mutation Gly679 → Ala had little effect on the kinetic properties of the enzyme. Compared with the wild-type enzyme, however, the Gly534 → Ala mutant exhibited a 4000-fold decrease in k cat and the Gly862 → Ala mutant showed an 11-fold increase inK m for ATP. Glucose 6-phosphate inhibition of the three glycine mutants is comparable to that of the wild-type enzyme. Inorganic phosphate is, however, less effective in relieving glucose 6-phosphate inhibition of the Gly862 → Ala mutant, relative to the wild-type enzyme and entirely ineffective in relieving inhibition of the Gly534 → Ala mutant. Although the fluorescence emission spectra showed some difference for the Gly862 → Ala mutant relative to that of the wild-type enzyme, indicating an environmental alteration around tryptophan residues, no change was observed for the Gly534 → Ala and Gly679 → Ala mutants. Gly862 → Ala and Gly534 → Ala are the first instances of single residue mutations in hexokinase I that affect the binding affinity of ATP and abolish phosphate-induced relief of glucose 6-phosphate inhibition, respectively. Mutants of hexokinase I (Arg539→ Lys, Thr661 → Ala, Thr661 → Val, Gly534 → Ala, Gly679 → Ala, and Gly862 → Ala), located putatively in the vicinity of the ATP binding pocket, were constructed, purified to homogeneity, and studied by circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and initial velocity kinetics. The wild-type and mutant enzymes have similar secondary structures on the basis of CD spectroscopy. The mutation Gly679 → Ala had little effect on the kinetic properties of the enzyme. Compared with the wild-type enzyme, however, the Gly534 → Ala mutant exhibited a 4000-fold decrease in k cat and the Gly862 → Ala mutant showed an 11-fold increase inK m for ATP. Glucose 6-phosphate inhibition of the three glycine mutants is comparable to that of the wild-type enzyme. Inorganic phosphate is, however, less effective in relieving glucose 6-phosphate inhibition of the Gly862 → Ala mutant, relative to the wild-type enzyme and entirely ineffective in relieving inhibition of the Gly534 → Ala mutant. Although the fluorescence emission spectra showed some difference for the Gly862 → Ala mutant relative to that of the wild-type enzyme, indicating an environmental alteration around tryptophan residues, no change was observed for the Gly534 → Ala and Gly679 → Ala mutants. Gly862 → Ala and Gly534 → Ala are the first instances of single residue mutations in hexokinase I that affect the binding affinity of ATP and abolish phosphate-induced relief of glucose 6-phosphate inhibition, respectively. Hexokinase catalyzes the phosphorylation of glucose, using ATP as a phosphoryl donor. Four isoforms of hexokinase exist in mammalian tissue (1Katzen H.M. Schimke R.T. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 1218-1225Crossref PubMed Scopus (272) Google Scholar). Hexokinase isoforms I, II, and III have molecular weights of approximately 100,000 and are monomers under most conditions. Amino acid sequences of isoforms I–III are 70% identical (2Wilson J.E. Rev. Physiol. Biochem. Pharmacol. 1995; 126: 65-198Crossref PubMed Google Scholar). Moreover the N- and C-terminal halves of isoforms I–III have similar amino acid sequences, probably as a result of gene duplication and fusion (3Easterby J.S. O'Brien M.J. Eur. J. Biochem. 1973; 38: 201-211Crossref PubMed Scopus (97) Google Scholar, 4Rose I.A. Warms J.V.B. Kosow D.P. Arch. Biochem. Biophys. 1974; 164: 729-735Crossref PubMed Scopus (60) Google Scholar, 5Holroyde M.J. Trayer I.P. FEBS Lett. 1976; 62: 215-219Crossref PubMed Scopus (60) Google Scholar, 6Ureta T. Comp. Biochem. Physiol. 1982; 71B: 549-555Google Scholar, 7Manning T.A. Wilson J.E. Biochem. Biophys. Res. Commun. 1984; 118: 90-96Crossref PubMed Scopus (14) Google Scholar). Hexokinase isoform IV (glucokinase) has a molecular weight of 50,000, similar to that of yeast hexokinase. Glucokinase exhibits (as does yeast hexokinase) significant sequence similarity to the N- and C-terminal halves of isoforms I–III.Despite sequence similarities, the functional properties of hexokinase isoforms differ significantly. Isoform I (hereafter, brain hexokinase or hexokinase I) governs the rate-limiting step of glycolysis in brain and red blood cells (8Lowry O.H. Passonneau J.V. J. Biol. Chem. 1964; 239: 31-42Abstract Full Text PDF PubMed Google Scholar, 9Rapoport S. Essays Biochem. 1968; 4: 69-103PubMed Google Scholar). The reaction product, glucose 6-phosphate (Glu-6-P 1The abbreviation used is: Glu-6-P, glucose 6-phosphate. 1The abbreviation used is: Glu-6-P, glucose 6-phosphate.), inhibits both isoforms I and II (but not isoform IV) at micromolar levels. Inorganic phosphate (Pi), however, reverses Glu-6-P inhibition of only hexokinase I. The C-terminal domain of hexokinase I possesses catalytic activity, whereas the N-terminal domain is involved in the Pi-induced relief of product inhibition (10White T.K. Wilson J.E. Arch. Biochem. Biophys. 1989; 274: 375-393Crossref PubMed Scopus (77) Google Scholar). In contrast, both the C- and N-terminal halves exhibit comparable catalytic activity in isoform II (11Tsai H.J. Wilson J.E. Arch. Biochem. Biophys. 1996; 329: 17-23Crossref PubMed Scopus (73) Google Scholar). Thus, among hexokinase isoforms, brain hexokinase exhibits unique regulatory properties in that physiological levels of Pi can reverse inhibition due to physiological levels of Glu-6-P (12Rudolph F.B. Fromm H.J. J. Biol. Chem. 1971; 246: 6611-6619Abstract Full Text PDF PubMed Google Scholar, 13Fromm H.J. Veneziale C.M. The Regulation of Carbohydrate Formation and Utilization in Mammals. University Park Press, Baltimore, MD1981: 45-68Google Scholar, 14Ureta T. Markert C.L. Isozymes III. Academic Press, Inc., New York1975: 575-601Google Scholar).The crystal structures of yeast hexokinase (15Anderson C.M. Stenkamp R.E. McDonald R.C. Steitz T.A. J. Mol. Biol. 1978; 123: 207-219Crossref PubMed Scopus (117) Google Scholar, 16Bennett Jr., W.S. Steitz T.A. J. Mol. Biol. 1980; 140: 211-230Crossref PubMed Scopus (205) Google Scholar, 17.Harrison, R., Crystallographic Refinement of Two Isozymes of Yeast Hexokinase and Relationship of Structure to Function.Ph.D. Thesis, 1985, Yale University, New Haven, CT.Google Scholar) are the basis for a model of mammalian glucokinase and its glucose binding site (18Charles R.S. Harrison R.W. Bell G.I. Pilkis S.J. Weber I.T. Diabetes. 1994; 43: 784-791Crossref PubMed Scopus (42) Google Scholar). The C-terminal domain of human brain hexokinase and its ATP binding site has been modeled in our laboratory based on similarities among the ATP-binding domains of actin, heat shock protein, and glycerol kinase (19Zeng C. Aleshin A.E. Hardie J.B. Harrison R.W. Fromm H.J. Biochemistry. 1996; 35: 13157-13164Crossref PubMed Scopus (42) Google Scholar). The model for the complex of ATP with hexokinase I puts a number of residues in the vicinity of ATP, of which Thr680, Asp532, and Arg539 have been the focus of directed mutations and investigations of initial rate kinetics (19Zeng C. Aleshin A.E. Hardie J.B. Harrison R.W. Fromm H.J. Biochemistry. 1996; 35: 13157-13164Crossref PubMed Scopus (42) Google Scholar,20Zeng C. Fromm H.J. J. Biol. Chem. 1995; 270: 10509-10513Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). These residues evidently stabilize the transition state, but do not influence the binding affinity of ATP (19Zeng C. Aleshin A.E. Hardie J.B. Harrison R.W. Fromm H.J. Biochemistry. 1996; 35: 13157-13164Crossref PubMed Scopus (42) Google Scholar, 20Zeng C. Fromm H.J. J. Biol. Chem. 1995; 270: 10509-10513Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar).This study presents the results of CD, fluorescence, and kinetic investigations of Gly534 → Ala, Arg539 → Lys, Thr661 → Ala, Thr661 → Val, Gly679 → Ala, and Gly862 → Ala mutants of brain hexokinase. The mutation Gly862 → Ala causes an order of magnitude increase in the K m for ATP, the first instance of a mutation in hexokinase I that has had a significant influence on the binding affinity of ATP. The mutation Gly534 → Ala reduces k cat by three orders of magnitude, but more significantly abolishes Pi-induced relief of Glu-6-P inhibition. Mutation at Gly534 is the first instance whereby a single mutation in hexokinase I has had a significant impact on the amelioration of product inhibition by Pi.DISCUSSIONArg539 putatively interacts with the polyphosphoryl portion of ATP and stabilizes the transition state (19Zeng C. Aleshin A.E. Hardie J.B. Harrison R.W. Fromm H.J. Biochemistry. 1996; 35: 13157-13164Crossref PubMed Scopus (42) Google Scholar, 20Zeng C. Fromm H.J. J. Biol. Chem. 1995; 270: 10509-10513Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The Arg539 → Lys mutant is 10-fold more active than the Arg539 → Ile mutant (20Zeng C. Fromm H.J. J. Biol. Chem. 1995; 270: 10509-10513Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), suggesting the importance of the positive charge at position 539. However, as the Arg539→ Lys mutant reported here is still 12-fold less active than wild-type hexokinase I, specific hydrogen bond interactions of the arginyl side chain are of equal importance in stabilizing the transition state. We have suggested, on the basis of previous work, that Arg539 may form salt bridges with oxygen atoms of the α- and β-phosphoryl groups of ATP (19Zeng C. Aleshin A.E. Hardie J.B. Harrison R.W. Fromm H.J. Biochemistry. 1996; 35: 13157-13164Crossref PubMed Scopus (42) Google Scholar). The observed properties of the Lys539 mutant are consistent with that suggestion.Thr661 is 10 Å away from the β-phosphoryl group of ATP in our model, however, the γ-oxygen atom of Thr661 is 3 Å from Asp657, which is putatively the catalytic base in the abstraction of a proton from the 6-hydroxyl group of glucose (25Arora K.K. Filburn C.R. Pedersen P.L. J. Biol. Chem. 1991; 266: 5359-5362Abstract Full Text PDF PubMed Google Scholar). The Thr661 → Ala mutant has kinetic properties similar to those of the wild-type enzyme, but mutation of Thr661 to valine causes a 9-fold decrease in k cat, probably by introducing an unfavorable nonbonded contact that perturbs Asp657.Consensus sequences, Gly-X-X-Gly-X-Gly-Lys-(Ser/Thr) in mononucleotide-binding proteins (26Bossemeyer D. Trends Biochem. Sci. 1994; 19: 201-205Abstract Full Text PDF PubMed Scopus (151) Google Scholar), Gly-X-Gly-X-X-Gly in dinucleotide-binding proteins (26Bossemeyer D. Trends Biochem. Sci. 1994; 19: 201-205Abstract Full Text PDF PubMed Scopus (151) Google Scholar), andY-Gly-X-Gly-X-(Phe/Tyr)-Gly-X-Val, where Y is a hydrophobic residue for protein kinases (27Hanks S.K. Quinn A.M. Methods Enzymol. 1991; 200: 38-62Crossref PubMed Scopus (1079) Google Scholar), are rich in conserved glycines. In the mononucleotide-binding protein, p21H-ras, the dihedral angles of the polypeptide chain require glycine (28Pai E.F. Krengel U. Petsko G.A. Goody R.S. Kabsch W. Wittinghofer A. EMBO J. 1990; 9: 2351-2359Crossref PubMed Scopus (958) Google Scholar). For dinucleotide-binding proteins (29Schulz G.E. Curr. Opin. Struct. Biol. 1992; 2: 61-67Crossref Scopus (299) Google Scholar), the second glycine of the consensus sequence provides space for the polyphosphoryl moiety, and the first and third glycines satisfy conformational constraints of the polypeptide chain. The glycine-rich sequences of protein kinases participate in nucleotide binding, substrate recognition, and enzyme catalysis (30Bossemeyer D. Nature. 1993; 363: 590Crossref PubMed Scopus (10) Google Scholar, 31Bossemeyer D. Engh R.A. Kinzel V. Ponstingl H. Huber R. EMBO J. 1993; 12: 849-859Crossref PubMed Scopus (371) Google Scholar). Yeast hexokinase, actin, hsc70, and glycerol kinase, however, are without a consensus sequence for nucleotide binding. Instead, the residues associated with nucleotide binding are scattered throughout the primary structure, but come together at single sites in the context of the folded polypeptide chains (32Bork P. Sander C. Valencia A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7290-7294Crossref PubMed Scopus (697) Google Scholar). Interestingly, the ATP binding domains of yeast hexokinase, actin, hsc70, and glycerol kinase are also rich in glycine residues.We have probed the corresponding glycines by directed mutation, in the expectation that some of these glycines are linked to observed kinetic properties in hexokinase I. The mutation of Gly534 to alanine produced a dramatic effect on k cat(4000-fold reduction) and modest effects on K m for glucose and ATP. Gly534 is conserved in sequences of hexokinase, but according to our model (Fig.3), its main chain torsion angles fall in the allowed region of the Ramachandran plot for alanine. Instead, Cβ of Ala534 is 3.6 Å from an oxygen of the β-phosphoryl group of ATP, but perhaps of greater significance is its 2.4 Å contact with backbone carbonyl 537 of an adjacent β-strand. Our model suggests then, the possibility of conformational change in the vicinity of residue 534 to relieve the close contact mentioned above. Such a local conformational change could influence Asp532, which on the basis of earlier work (19Zeng C. Aleshin A.E. Hardie J.B. Harrison R.W. Fromm H.J. Biochemistry. 1996; 35: 13157-13164Crossref PubMed Scopus (42) Google Scholar) plays a critical role in the stabilization of the transition state and may be involved in the binding of Mg2+. A larger perturbation on the active site due to the mutation of Gly534 to alanine is not likely, because K m values for substrates are not influenced and CD spectroscopy indicates no change in secondary structure.The Gly534 → Ala mutant represents the first instance whereby the mutation of a single residue has abolished Pi-induced relief of Glu-6-P inhibition in hexokinase I. Glu-6-P inhibition of the C-terminal half of hexokinase I (mini-hexokinase) cannot be reversed by Pi, implicating the N-terminal domain in the relief of inhibition (20Zeng C. Fromm H.J. J. Biol. Chem. 1995; 270: 10509-10513Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The mechanism by which Pi relieves Glu-6-P inhibition then evidently involves structural elements of both the N- and C-terminal halves of hexokinase I. Furthermore, the loss of Pi-induced relief of Glu-6-P inhibition in the Gly534 → Ala mutant is linked closely to position 534, as mutations of Asp532 to lysine and glutamate have little effect on this property (19Zeng C. Aleshin A.E. Hardie J.B. Harrison R.W. Fromm H.J. Biochemistry. 1996; 35: 13157-13164Crossref PubMed Scopus (42) Google Scholar).Gly679 and Gly862 belong to reverse turns, which pack against each other in our model (Fig. 3). The main chain torsion angles put positions 679 and 862 in unallowed regions of the Ramachandran plot for alanine (φ = −111, ψ = −133 for Gly679; φ = 70, ψ = 168 for Gly862). Of the two mutants, Gly862 → Ala has the conformation of highest energy. Although the Cβ atoms at positions 679 and 862 probably do not interact with ATP, they make unfavorable contacts in our model with backbone amide 863 (2.6 Å) and backbone amide 679 (2.7 Å), respectively. These unfavorable contacts may not be significant, however, as the mutation of Gly679 to alanine has no effect on the kinetic properties of the enzyme. Instead, the introduction of alanine at position 862 probably causes conformational changes in main chain torsion angles. Although the fluorescence spectra of the wild-type and Gly862 → Ala enzymes differ (indicating a perturbation in the local environment of tryptophan residues) their CD spectra are identical (indicating no change in secondary structure). Furthermore, the K i for 1,5-anhydroglucitol-6-phosphate and the K m for glucose are similar for the Gly682 → Ala mutant and the wild-type enzyme. Thus the mutation of Gly862 to alanine probably has an effect only on residues in the vicinity of position 862. Thr863, a residue conserved in hexokinase sequences, interacts with the ribose and base moieties of ATP in our model (Fig.3). The Gly682 → Ala mutant, then, could influence interactions involving the base of ATP by perturbing the conformation or relative position of Thr683. The mutation of Gly862 to alanine increases the K m for ATP by 11-fold without large changes in other kinetic parameters and as such, represents the first mutation, which to our knowledge influences the binding affinity of ATP.Mutations prepared here and from previous studies (19Zeng C. Aleshin A.E. Hardie J.B. Harrison R.W. Fromm H.J. Biochemistry. 1996; 35: 13157-13164Crossref PubMed Scopus (42) Google Scholar) show a trend that may be significant to the function of hexokinases in general. Mutations of hexokinase I, which putatively influence interactions involving the polyphosphoryl moiety of ATP, have no effect onK m but a large effect onk cat. Conversely, the Gly862 → Ala mutant, which putatively influences interactions at the base moiety, has little effect on k cat but substantial effects on K m for ATP. Interactions involving the base of ATP are important for affinity, but polyphosphoryl-protein interactions are involved in the stabilization of the transition state. Conceivably, hexokinase I diverts energy from favorable interactions between the polyphosphoryl moiety of ATP and the enzyme to promote conformational changes that stabilize the transition state. This phenomenon is not without precedence. In adenylosuccinate synthetase from E. coli mutations involving protein interactions at the base of GTP affect K m (33Kang C. Sun N. Honzatko R.B. Fromm H.J. J. Biol. Chem. 1994; 269: 24046-24049Abstract Full Text PDF PubMed Google Scholar), whereas interactions between the polyphosphoryl group of GTP and the protein (and Mg2+) contribute to the stability of the transition state by driving conformational changes in the active site (34Poland B.W. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1996; 264: 1013-1027Crossref PubMed Scopus (44) Google Scholar). Hexokinase catalyzes the phosphorylation of glucose, using ATP as a phosphoryl donor. Four isoforms of hexokinase exist in mammalian tissue (1Katzen H.M. Schimke R.T. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 1218-1225Crossref PubMed Scopus (272) Google Scholar). Hexokinase isoforms I, II, and III have molecular weights of approximately 100,000 and are monomers under most conditions. Amino acid sequences of isoforms I–III are 70% identical (2Wilson J.E. Rev. Physiol. Biochem. Pharmacol. 1995; 126: 65-198Crossref PubMed Google Scholar). Moreover the N- and C-terminal halves of isoforms I–III have similar amino acid sequences, probably as a result of gene duplication and fusion (3Easterby J.S. O'Brien M.J. Eur. J. Biochem. 1973; 38: 201-211Crossref PubMed Scopus (97) Google Scholar, 4Rose I.A. Warms J.V.B. Kosow D.P. Arch. Biochem. Biophys. 1974; 164: 729-735Crossref PubMed Scopus (60) Google Scholar, 5Holroyde M.J. Trayer I.P. FEBS Lett. 1976; 62: 215-219Crossref PubMed Scopus (60) Google Scholar, 6Ureta T. Comp. Biochem. Physiol. 1982; 71B: 549-555Google Scholar, 7Manning T.A. Wilson J.E. Biochem. Biophys. Res. Commun. 1984; 118: 90-96Crossref PubMed Scopus (14) Google Scholar). Hexokinase isoform IV (glucokinase) has a molecular weight of 50,000, similar to that of yeast hexokinase. Glucokinase exhibits (as does yeast hexokinase) significant sequence similarity to the N- and C-terminal halves of isoforms I–III. Despite sequence similarities, the functional properties of hexokinase isoforms differ significantly. Isoform I (hereafter, brain hexokinase or hexokinase I) governs the rate-limiting step of glycolysis in brain and red blood cells (8Lowry O.H. Passonneau J.V. J. Biol. Chem. 1964; 239: 31-42Abstract Full Text PDF PubMed Google Scholar, 9Rapoport S. Essays Biochem. 1968; 4: 69-103PubMed Google Scholar). The reaction product, glucose 6-phosphate (Glu-6-P 1The abbreviation used is: Glu-6-P, glucose 6-phosphate. 1The abbreviation used is: Glu-6-P, glucose 6-phosphate.), inhibits both isoforms I and II (but not isoform IV) at micromolar levels. Inorganic phosphate (Pi), however, reverses Glu-6-P inhibition of only hexokinase I. The C-terminal domain of hexokinase I possesses catalytic activity, whereas the N-terminal domain is involved in the Pi-induced relief of product inhibition (10White T.K. Wilson J.E. Arch. Biochem. Biophys. 1989; 274: 375-393Crossref PubMed Scopus (77) Google Scholar). In contrast, both the C- and N-terminal halves exhibit comparable catalytic activity in isoform II (11Tsai H.J. Wilson J.E. Arch. Biochem. Biophys. 1996; 329: 17-23Crossref PubMed Scopus (73) Google Scholar). Thus, among hexokinase isoforms, brain hexokinase exhibits unique regulatory properties in that physiological levels of Pi can reverse inhibition due to physiological levels of Glu-6-P (12Rudolph F.B. Fromm H.J. J. Biol. Chem. 1971; 246: 6611-6619Abstract Full Text PDF PubMed Google Scholar, 13Fromm H.J. Veneziale C.M. The Regulation of Carbohydrate Formation and Utilization in Mammals. University Park Press, Baltimore, MD1981: 45-68Google Scholar, 14Ureta T. Markert C.L. Isozymes III. Academic Press, Inc., New York1975: 575-601Google Scholar). The crystal structures of yeast hexokinase (15Anderson C.M. Stenkamp R.E. McDonald R.C. Steitz T.A. J. Mol. Biol. 1978; 123: 207-219Crossref PubMed Scopus (117) Google Scholar, 16Bennett Jr., W.S. Steitz T.A. J. Mol. Biol. 1980; 140: 211-230Crossref PubMed Scopus (205) Google Scholar, 17.Harrison, R., Crystallographic Refinement of Two Isozymes of Yeast Hexokinase and Relationship of Structure to Function.Ph.D. Thesis, 1985, Yale University, New Haven, CT.Google Scholar) are the basis for a model of mammalian glucokinase and its glucose binding site (18Charles R.S. Harrison R.W. Bell G.I. Pilkis S.J. Weber I.T. Diabetes. 1994; 43: 784-791Crossref PubMed Scopus (42) Google Scholar). The C-terminal domain of human brain hexokinase and its ATP binding site has been modeled in our laboratory based on similarities among the ATP-binding domains of actin, heat shock protein, and glycerol kinase (19Zeng C. Aleshin A.E. Hardie J.B. Harrison R.W. Fromm H.J. Biochemistry. 1996; 35: 13157-13164Crossref PubMed Scopus (42) Google Scholar). The model for the complex of ATP with hexokinase I puts a number of residues in the vicinity of ATP, of which Thr680, Asp532, and Arg539 have been the focus of directed mutations and investigations of initial rate kinetics (19Zeng C. Aleshin A.E. Hardie J.B. Harrison R.W. Fromm H.J. Biochemistry. 1996; 35: 13157-13164Crossref PubMed Scopus (42) Google Scholar,20Zeng C. Fromm H.J. J. Biol. Chem. 1995; 270: 10509-10513Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). These residues evidently stabilize the transition state, but do not influence the binding affinity of ATP (19Zeng C. Aleshin A.E. Hardie J.B. Harrison R.W. Fromm H.J. Biochemistry. 1996; 35: 13157-13164Crossref PubMed Scopus (42) Google Scholar, 20Zeng C. Fromm H.J. J. Biol. Chem. 1995; 270: 10509-10513Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). This study presents the results of CD, fluorescence, and kinetic investigations of Gly534 → Ala, Arg539 → Lys, Thr661 → Ala, Thr661 → Val, Gly679 → Ala, and Gly862 → Ala mutants of brain hexokinase. The mutation Gly862 → Ala causes an order of magnitude increase in the K m for ATP, the first instance of a mutation in hexokinase I that has had a significant influence on the binding affinity of ATP. The mutation Gly534 → Ala reduces k cat by three orders of magnitude, but more significantly abolishes Pi-induced relief of Glu-6-P inhibition. Mutation at Gly534 is the first instance whereby a single mutation in hexokinase I has had a significant impact on the amelioration of product inhibition by Pi. DISCUSSIONArg539 putatively interacts with the polyphosphoryl portion of ATP and stabilizes the transition state (19Zeng C. Aleshin A.E. Hardie J.B. Harrison R.W. Fromm H.J. Biochemistry. 1996; 35: 13157-13164Crossref PubMed Scopus (42) Google Scholar, 20Zeng C. Fromm H.J. J. Biol. Chem. 1995; 270: 10509-10513Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The Arg539 → Lys mutant is 10-fold more active than the Arg539 → Ile mutant (20Zeng C. Fromm H.J. J. Biol. Chem. 1995; 270: 10509-10513Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), suggesting the importance of the positive charge at position 539. However, as the Arg539→ Lys mutant reported here is still 12-fold less active than wild-type hexokinase I, specific hydrogen bond interactions of the arginyl side chain are of equal importance in stabilizing the transition state. We have suggested, on the basis of previous work, that Arg539 may form salt bridges with oxygen atoms of the α- and β-phosphoryl groups of ATP (19Zeng C. Aleshin A.E. Hardie J.B. Harrison R.W. Fromm H.J. Biochemistry. 1996; 35: 13157-13164Crossref PubMed Scopus (42) Google Scholar). The observed properties of the Lys539 mutant are consistent with that suggestion.Thr661 is 10 Å away from the β-phosphoryl group of ATP in our model, however, the γ-oxygen atom of Thr661 is 3 Å from Asp657, which is putatively the catalytic base in the abstraction of a proton from the 6-hydroxyl group of glucose (25Arora K.K. Filburn C.R. Pedersen P.L. J. Biol. Chem. 1991; 266: 5359-5362Abstract Full Text PDF PubMed Google Scholar). The Thr661 → Ala mutant has kinetic properties similar to those of the wild-type enzyme, but mutation of Thr661 to valine causes a 9-fold decrease in k cat, probably by introducing an unfavorable nonbonded contact that perturbs Asp657.Consensus sequences, Gly-X-X-Gly-X-Gly-Lys-(Ser/Thr) in mononucleotide-binding proteins (26Bossemeyer D. Trends Biochem. Sci. 1994; 19: 201-205Abstract Full Text PDF PubMed Scopus (151) Google Scholar), Gly-X-Gly-X-X-Gly in dinucleotide-binding proteins (26Bossemeyer D. Trends Biochem. Sci. 1994; 19: 201-205Abstract Full Text PDF PubMed Scopus (151) Google Scholar), andY-Gly-X-Gly-X-(Phe/Tyr)-Gly-X-Val, where Y is a hydrophobic residue for protein kinases (27Hanks S.K. Quinn A.M. Methods Enzymol. 1991; 200: 38-62Crossref PubMed Scopus (1079) Google Scholar), are rich in conserved glycines. In the mononucleotide-binding protein, p21H-ras, the dihedral angles of the polypeptide chain require glycine (28Pai E.F. Krengel U. Petsko G.A. Goody R.S. Kabsch W. Wittinghofer A. EMBO J. 1990; 9: 2351-2359Crossref PubMed Scopus (958) Google Scholar). For dinucleotide-binding proteins (29Schulz G.E. Curr. Opin. Struct. Biol. 1992; 2: 61-67Crossref Scopus (299) Google Scholar), the second glycine of the consensus sequence provides space for the polyphosphoryl moiety, and the first and third glycines satisfy conformational constraints of the polypeptide chain. The glycine-rich sequences of protein kinases participate in nucleotide binding, substrate recognition, and enzyme catalysis (30Bossemeyer D. Nature. 1993; 363: 590Crossref PubMed Scopus (10) Google Scholar, 31Bossemeyer D. Engh R.A. Kinzel V. Ponstingl H. Huber R. EMBO J. 1993; 12: 849-859Crossref PubMed Scopus (371) Google Scholar). Yeast hexokinase, actin, hsc70, and glycerol kinase, however, are without a consensus sequence for nucleotide binding. Instead, the residues associated with nucleotide binding are scattered throughout the primary structure, but come together at single sites in the context of the folded polypeptide chains (32Bork P. Sander C. Valencia A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7290-7294Crossref PubMed Scopus (697) Google Scholar). Interestingly, the ATP binding domains of yeast hexokinase, actin, hsc70, and glycerol kinase are also rich in glycine residues.We have probed the corresponding glycines by directed mutation, in the expectation that some of these glycines are linked to observed kinetic properties in hexokinase I. The mutation of Gly534 to alanine produced a dramatic effect on k cat(4000-fold reduction) and modest effects on K m for glucose and ATP. Gly534 is conserved in sequences of hexokinase, but according to our model (Fig.3), its main chain torsion angles fall in the allowed region of the Ramachandran plot for alanine. Instead, Cβ of Ala534 is 3.6 Å from an oxygen of the β-phosphoryl group of ATP, but perhaps of greater significance is its 2.4 Å contact with backbone carbonyl 537 of an adjacent β-strand. Our model suggests then, the possibility of conformational change in the vicinity of residue 534 to relieve the close contact mentioned above. Such a local conformational change could influence Asp532, which on the basis of earlier work (19Zeng C. Aleshin A.E. Hardie J.B. Harrison R.W. Fromm H.J. Biochemistry. 1996; 35: 13157-13164Crossref PubMed Scopus (42) Google Scholar) plays a critical role in the stabilization of the transition state and may be involved in the binding of Mg2+. A larger perturbation on the active site due to the mutation of Gly534 to alanine is not likely, because K m values for substrates are not influenced and CD spectroscopy indicates no change in secondary structure.The Gly534 → Ala mutant represents the first instance whereby the mutation of a single residue has abolished Pi-induced relief of Glu-6-P inhibition in hexokinase I. Glu-6-P inhibition of the C-terminal half of hexokinase I (mini-hexokinase) cannot be reversed by Pi, implicating the N-terminal domain in the relief of inhibition (20Zeng C. Fromm H.J. J. Biol. Chem. 1995; 270: 10509-10513Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The mechanism by which Pi relieves Glu-6-P inhibition then evidently involves structural elements of both the N- and C-terminal halves of hexokinase I. Furthermore, the loss of Pi-induced relief of Glu-6-P inhibition in the Gly534 → Ala mutant is linked closely to position 534, as mutations of Asp532 to lysine and glutamate have little effect on this property (19Zeng C. Aleshin A.E. Hardie J.B. Harrison R.W. Fromm H.J. Biochemistry. 1996; 35: 13157-13164Crossref PubMed Scopus (42) Google Scholar).Gly679 and Gly862 belong to reverse turns, which pack against each other in our model (Fig. 3). The main chain torsion angles put positions 679 and 862 in unallowed regions of the Ramachandran plot for alanine (φ = −111, ψ = −133 for Gly679; φ = 70, ψ = 168 for Gly862). Of the two mutants, Gly862 → Ala has the conformation of highest energy. Although the Cβ atoms at positions 679 and 862 probably do not interact with ATP, they make unfavorable contacts in our model with backbone amide 863 (2.6 Å) and backbone amide 679 (2.7 Å), respectively. These unfavorable contacts may not be significant, however, as the mutation of Gly679 to alanine has no effect on the kinetic properties of the enzyme. Instead, the introduction of alanine at position 862 probably causes conformational changes in main chain torsion angles. Although the fluorescence spectra of the wild-type and Gly862 → Ala enzymes differ (indicating a perturbation in the local environment of tryptophan residues) their CD spectra are identical (indicating no change in secondary structure). Furthermore, the K i for 1,5-anhydroglucitol-6-phosphate and the K m for glucose are similar for the Gly682 → Ala mutant and the wild-type enzyme. Thus the mutation of Gly862 to alanine probably has an effect only on residues in the vicinity of position 862. Thr863, a residue conserved in hexokinase sequences, interacts with the ribose and base moieties of ATP in our model (Fig.3). The Gly682 → Ala mutant, then, could influence interactions involving the base of ATP by perturbing the conformation or relative position of Thr683. The mutation of Gly862 to alanine increases the K m for ATP by 11-fold without large changes in other kinetic parameters and as such, represents the first mutation, which to our knowledge influences the binding affinity of ATP.Mutations prepared here and from previous studies (19Zeng C. Aleshin A.E. Hardie J.B. Harrison R.W. Fromm H.J. Biochemistry. 1996; 35: 13157-13164Crossref PubMed Scopus (42) Google Scholar) show a trend that may be significant to the function of hexokinases in general. Mutations of hexokinase I, which putatively influence interactions involving the polyphosphoryl moiety of ATP, have no effect onK m but a large effect onk cat. Conversely, the Gly862 → Ala mutant, which putatively influences interactions at the base moiety, has little effect on k cat but substantial effects on K m for ATP. Interactions involving the base of ATP are important for affinity, but polyphosphoryl-protein interactions are involved in the stabilization of the transition state. Conceivably, hexokinase I diverts energy from favorable interactions between the polyphosphoryl moiety of ATP and the enzyme to promote conformational changes that stabilize the transition state. This phenomenon is not without precedence. In adenylosuccinate synthetase from E. coli mutations involving protein interactions at the base of GTP affect K m (33Kang C. Sun N. Honzatko R.B. Fromm H.J. J. Biol. Chem. 1994; 269: 24046-24049Abstract Full Text PDF PubMed Google Scholar), whereas interactions between the polyphosphoryl group of GTP and the protein (and Mg2+) contribute to the stability of the transition state by driving conformational changes in the active site (34Poland B.W. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1996; 264: 1013-1027Crossref PubMed Scopus (44) Google Scholar). Arg539 putatively interacts with the polyphosphoryl portion of ATP and stabilizes the transition state (19Zeng C. Aleshin A.E. Hardie J.B. Harrison R.W. Fromm H.J. Biochemistry. 1996; 35: 13157-13164Crossref PubMed Scopus (42) Google Scholar, 20Zeng C. Fromm H.J. J. Biol. Chem. 1995; 270: 10509-10513Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The Arg539 → Lys mutant is 10-fold more active than the Arg539 → Ile mutant (20Zeng C. Fromm H.J. J. Biol. Chem. 1995; 270: 10509-10513Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), suggesting the importance of the positive charge at position 539. However, as the Arg539→ Lys mutant reported here is still 12-fold less active than wild-type hexokinase I, specific hydrogen bond interactions of the arginyl side chain are of equal importance in stabilizing the transition state. We have suggested, on the basis of previous work, that Arg539 may form salt bridges with oxygen atoms of the α- and β-phosphoryl groups of ATP (19Zeng C. Aleshin A.E. Hardie J.B. Harrison R.W. Fromm H.J. Biochemistry. 1996; 35: 13157-13164Crossref PubMed Scopus (42) Google Scholar). The observed properties of the Lys539 mutant are consistent with that suggestion. Thr661 is 10 Å away from the β-phosphoryl group of ATP in our model, however, the γ-oxygen atom of Thr661 is 3 Å from Asp657, which is putatively the catalytic base in the abstraction of a proton from the 6-hydroxyl group of glucose (25Arora K.K. Filburn C.R. Pedersen P.L. J. Biol. Chem. 1991; 266: 5359-5362Abstract Full Text PDF PubMed Google Scholar). The Thr661 → Ala mutant has kinetic properties similar to those of the wild-type enzyme, but mutation of Thr661 to valine causes a 9-fold decrease in k cat, probably by introducing an unfavorable nonbonded contact that perturbs Asp657. Consensus sequences, Gly-X-X-Gly-X-Gly-Lys-(Ser/Thr) in mononucleotide-binding proteins (26Bossemeyer D. Trends Biochem. Sci. 1994; 19: 201-205Abstract Full Text PDF PubMed Scopus (151) Google Scholar), Gly-X-Gly-X-X-Gly in dinucleotide-binding proteins (26Bossemeyer D. Trends Biochem. Sci. 1994; 19: 201-205Abstract Full Text PDF PubMed Scopus (151) Google Scholar), andY-Gly-X-Gly-X-(Phe/Tyr)-Gly-X-Val, where Y is a hydrophobic residue for protein kinases (27Hanks S.K. Quinn A.M. Methods Enzymol. 1991; 200: 38-62Crossref PubMed Scopus (1079) Google Scholar), are rich in conserved glycines. In the mononucleotide-binding protein, p21H-ras, the dihedral angles of the polypeptide chain require glycine (28Pai E.F. Krengel U. Petsko G.A. Goody R.S. Kabsch W. Wittinghofer A. EMBO J. 1990; 9: 2351-2359Crossref PubMed Scopus (958) Google Scholar). For dinucleotide-binding proteins (29Schulz G.E. Curr. Opin. Struct. Biol. 1992; 2: 61-67Crossref Scopus (299) Google Scholar), the second glycine of the consensus sequence provides space for the polyphosphoryl moiety, and the first and third glycines satisfy conformational constraints of the polypeptide chain. The glycine-rich sequences of protein kinases participate in nucleotide binding, substrate recognition, and enzyme catalysis (30Bossemeyer D. Nature. 1993; 363: 590Crossref PubMed Scopus (10) Google Scholar, 31Bossemeyer D. Engh R.A. Kinzel V. Ponstingl H. Huber R. EMBO J. 1993; 12: 849-859Crossref PubMed Scopus (371) Google Scholar). Yeast hexokinase, actin, hsc70, and glycerol kinase, however, are without a consensus sequence for nucleotide binding. Instead, the residues associated with nucleotide binding are scattered throughout the primary structure, but come together at single sites in the context of the folded polypeptide chains (32Bork P. Sander C. Valencia A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7290-7294Crossref PubMed Scopus (697) Google Scholar). Interestingly, the ATP binding domains of yeast hexokinase, actin, hsc70, and glycerol kinase are also rich in glycine residues. We have probed the corresponding glycines by directed mutation, in the expectation that some of these glycines are linked to observed kinetic properties in hexokinase I. The mutation of Gly534 to alanine produced a dramatic effect on k cat(4000-fold reduction) and modest effects on K m for glucose and ATP. Gly534 is conserved in sequences of hexokinase, but according to our model (Fig.3), its main chain torsion angles fall in the allowed region of the Ramachandran plot for alanine. Instead, Cβ of Ala534 is 3.6 Å from an oxygen of the β-phosphoryl group of ATP, but perhaps of greater significance is its 2.4 Å contact with backbone carbonyl 537 of an adjacent β-strand. Our model suggests then, the possibility of conformational change in the vicinity of residue 534 to relieve the close contact mentioned above. Such a local conformational change could influence Asp532, which on the basis of earlier work (19Zeng C. Aleshin A.E. Hardie J.B. Harrison R.W. Fromm H.J. Biochemistry. 1996; 35: 13157-13164Crossref PubMed Scopus (42) Google Scholar) plays a critical role in the stabilization of the transition state and may be involved in the binding of Mg2+. A larger perturbation on the active site due to the mutation of Gly534 to alanine is not likely, because K m values for substrates are not influenced and CD spectroscopy indicates no change in secondary structure. The Gly534 → Ala mutant represents the first instance whereby the mutation of a single residue has abolished Pi-induced relief of Glu-6-P inhibition in hexokinase I. Glu-6-P inhibition of the C-terminal half of hexokinase I (mini-hexokinase) cannot be reversed by Pi, implicating the N-terminal domain in the relief of inhibition (20Zeng C. Fromm H.J. J. Biol. Chem. 1995; 270: 10509-10513Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The mechanism by which Pi relieves Glu-6-P inhibition then evidently involves structural elements of both the N- and C-terminal halves of hexokinase I. Furthermore, the loss of Pi-induced relief of Glu-6-P inhibition in the Gly534 → Ala mutant is linked closely to position 534, as mutations of Asp532 to lysine and glutamate have little effect on this property (19Zeng C. Aleshin A.E. Hardie J.B. Harrison R.W. Fromm H.J. Biochemistry. 1996; 35: 13157-13164Crossref PubMed Scopus (42) Google Scholar). Gly679 and Gly862 belong to reverse turns, which pack against each other in our model (Fig. 3). The main chain torsion angles put positions 679 and 862 in unallowed regions of the Ramachandran plot for alanine (φ = −111, ψ = −133 for Gly679; φ = 70, ψ = 168 for Gly862). Of the two mutants, Gly862 → Ala has the conformation of highest energy. Although the Cβ atoms at positions 679 and 862 probably do not interact with ATP, they make unfavorable contacts in our model with backbone amide 863 (2.6 Å) and backbone amide 679 (2.7 Å), respectively. These unfavorable contacts may not be significant, however, as the mutation of Gly679 to alanine has no effect on the kinetic properties of the enzyme. Instead, the introduction of alanine at position 862 probably causes conformational changes in main chain torsion angles. Although the fluorescence spectra of the wild-type and Gly862 → Ala enzymes differ (indicating a perturbation in the local environment of tryptophan residues) their CD spectra are identical (indicating no change in secondary structure). Furthermore, the K i for 1,5-anhydroglucitol-6-phosphate and the K m for glucose are similar for the Gly682 → Ala mutant and the wild-type enzyme. Thus the mutation of Gly862 to alanine probably has an effect only on residues in the vicinity of position 862. Thr863, a residue conserved in hexokinase sequences, interacts with the ribose and base moieties of ATP in our model (Fig.3). The Gly682 → Ala mutant, then, could influence interactions involving the base of ATP by perturbing the conformation or relative position of Thr683. The mutation of Gly862 to alanine increases the K m for ATP by 11-fold without large changes in other kinetic parameters and as such, represents the first mutation, which to our knowledge influences the binding affinity of ATP. Mutations prepared here and from previous studies (19Zeng C. Aleshin A.E. Hardie J.B. Harrison R.W. Fromm H.J. Biochemistry. 1996; 35: 13157-13164Crossref PubMed Scopus (42) Google Scholar) show a trend that may be significant to the function of hexokinases in general. Mutations of hexokinase I, which putatively influence interactions involving the polyphosphoryl moiety of ATP, have no effect onK m but a large effect onk cat. Conversely, the Gly862 → Ala mutant, which putatively influences interactions at the base moiety, has little effect on k cat but substantial effects on K m for ATP. Interactions involving the base of ATP are important for affinity, but polyphosphoryl-protein interactions are involved in the stabilization of the transition state. Conceivably, hexokinase I diverts energy from favorable interactions between the polyphosphoryl moiety of ATP and the enzyme to promote conformational changes that stabilize the transition state. This phenomenon is not without precedence. In adenylosuccinate synthetase from E. coli mutations involving protein interactions at the base of GTP affect K m (33Kang C. Sun N. Honzatko R.B. Fromm H.J. J. Biol. Chem. 1994; 269: 24046-24049Abstract Full Text PDF PubMed Google Scholar), whereas interactions between the polyphosphoryl group of GTP and the protein (and Mg2+) contribute to the stability of the transition state by driving conformational changes in the active site (34Poland B.W. Fromm H.J. Honzatko R.B. J. Mol. Biol. 1996; 264: 1013-1027Crossref PubMed Scopus (44) Google Scholar). We thank Laura Duck for excellent technical assistance.
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