Effect of pH on the Stability and Structure of Yeast Hexokinase A

pH and salts have a marked effect on the stability, structure, and function of many globular proteins due to their ability to influence the electrostatic interactions. In this work, calorimetry, CD, and fluorescence studies have been carried out to understand the pH-dependent conformational changes of the two-domain protein yeast hexokinase A. In conjunction with the crystal structural data available, the present results have enabled the complete characterization and analysis of the pH-dependent conformational changes of the enzyme that have strong implications in understanding its structure-function relationship. The calorimetric profiles show a single thermal transition in the acidic pH range, whereas two independent transitions were observed in the alkaline pH range, suggesting the structural merger of the domains at the acidic pH. Comparison of the thermal transitions at pH 8.5 studied by different techniques suggests that the first transition corresponds to the smaller domain, and the second transition corresponds to the larger domain. The acid-denatured state of hexokinase A has high secondary structure content with little or no tertiary interactions and binds to the hydrophobic dye 8-anilinonaphthalene-1-sulfonic acid, suggesting that it is a molten globule-like state, whereas the alkali-denatured state is less structured than the acid-denatured state but more structured than the urea-denatured state, suggestive of a premolten globule-like state. Structural analysis using the published hexokinase B structure as well as the hexokinase A structure with the revised amino acid sequence in conjunction with the results obtained by us suggests that the ionization state of the acidic residues at the active site could regulate domain movements that are responsible for the opening and the closure of the cleft between the two domains and in turn affect the structure and function of the enzyme. pH and salts have a marked effect on the stability, structure, and function of many globular proteins due to their ability to influence the electrostatic interactions. In this work, calorimetry, CD, and fluorescence studies have been carried out to understand the pH-dependent conformational changes of the two-domain protein yeast hexokinase A. In conjunction with the crystal structural data available, the present results have enabled the complete characterization and analysis of the pH-dependent conformational changes of the enzyme that have strong implications in understanding its structure-function relationship. The calorimetric profiles show a single thermal transition in the acidic pH range, whereas two independent transitions were observed in the alkaline pH range, suggesting the structural merger of the domains at the acidic pH. Comparison of the thermal transitions at pH 8.5 studied by different techniques suggests that the first transition corresponds to the smaller domain, and the second transition corresponds to the larger domain. The acid-denatured state of hexokinase A has high secondary structure content with little or no tertiary interactions and binds to the hydrophobic dye 8-anilinonaphthalene-1-sulfonic acid, suggesting that it is a molten globule-like state, whereas the alkali-denatured state is less structured than the acid-denatured state but more structured than the urea-denatured state, suggestive of a premolten globule-like state. Structural analysis using the published hexokinase B structure as well as the hexokinase A structure with the revised amino acid sequence in conjunction with the results obtained by us suggests that the ionization state of the acidic residues at the active site could regulate domain movements that are responsible for the opening and the closure of the cleft between the two domains and in turn affect the structure and function of the enzyme. Hexokinase is a member of the kinase family of tissue-specific isozymes and is the first enzyme in the glycolytic pathway, catalyzing the transfer of a phosphoryl group from ATP to glucose to form glucose 6-phosphate and ADP with the release of a proton. Its malfunction has been implicated in a number of diseases in humans. Reduction in the activity of hexokinase due to mutations at the active site in humans causes hemolytic anemia (1Magnani M. Stocchi V. Cucchiarini L. Novelli G. Lodi S. Isa L. Fornaini G. Blood. 1985; 66: 690-697Google Scholar, 2Peters L.L. Lane P.W. Andersen S.G. Gwynn B. Barker J.E. Beutler E. Blood Cells Mol. Dis. 2001; 27: 850-860Google Scholar) and cardiomyopathy (3Barrie S.E. Saad E.A. Ubatuba S. Da Silva Lacaz P. Harris P. Res. Commun. Chem. Pathol. Pharmacol. 1979; 23: 375-381Google Scholar). Type II hexokinase-mitochondrial interactions were observed to promote tumor cell growth, and hexokinase has been suggested as the ideal target for therapeutic intervention (4Smith T.A. Br. J. Biomed. Sci. 2000; 57: 170-178Google Scholar, 5Pedersen P.L. Mathupala S. Rempel A. Geschwind J.F. Ko Y.H. Biochim. Biophys. Acta. 2002; 1555: 14-20Google Scholar). Modifications in the catalytic activity of hexokinase have been suggested to play a role in the pathogenesis of the Alzheimer's disease (6Sorbi S. Mortilla M. Piacentini S. Tonini S. Amaducci L. Neurosci. Lett. 1990; 117: 165-168Google Scholar). Recently, it has been shown that hexokinase plays a role in sensing and maintaining the glucose levels and in signal transduction to regulate the expression of genes in plants (7Moore B. Zhou L. Rolland F. Hall Q. Cheng W.H. Liu Y.X. Hwang I. Jones T. Sheen J. Science. 2003; 300: 332-336Google Scholar, 8Frommer W.B. Schulze W.X. Lalonde S. Science. 2003; 300: 261-263Google Scholar). In yeast, two isozymes named A and B (hexokinase PI and PII, respectively (9Womack F.C. Welch M.K. Nielsen J. Colowick S.P. Arch. Biochem. Biophys. 1973; 158: 451-457Google Scholar)) are known, with 76% overall homology of the amino acid sequence (10Kuser P.R. Krauchenco S. Antunes O.A. Polikarpov I. J. Biol. Chem. 2000; 275: 20814-20821Google Scholar). Hexokinase isozymes are known to exist as dimers of 100 kDa. Endogenous protease action during purification leads to the loss of 11 amino acids from the N terminus, resulting in a predominantly monomeric form of 50 kDa (11Colowick S.P. Boyer P.D. The Enzymes. 9. Academic Press, New York1973: 1-48Google Scholar, 12Schmidt J.J. Colowick S.P. Arch. Biochem. Biophys. 1973; 158: 458-470Google Scholar). The crystal structures are available for both of the hexokinase isozymes, for hexokinase PI complexed with glucose (13Bennett W.S.J. Steitz T.A. J. Mol. Biol. 1980; 140: 183-209Google Scholar, 14Bennett W.S.J. Steitz T.A. J. Mol. Biol. 1980; 140: 211-230Google Scholar) and for hexokinase PII complexed with ortho-toluoylglucosamine, a competitive inhibitor (15Steitz T.A. J. Mol. Biol. 1971; 61: 695-700Google Scholar). Both of these crystal structures, however, have missing residues, since at that time the amino acid sequence was not available, and the side chains were deduced from the electron density with only 30% of the amino acid sequence homology between the primary structure of the crystallographic models and the one obtained from cDNA sequence (10Kuser P.R. Krauchenco S. Antunes O.A. Polikarpov I. J. Biol. Chem. 2000; 275: 20814-20821Google Scholar). Recently, the PII structure without any bound ligands and with a correct amino acid sequence has become available (10Kuser P.R. Krauchenco S. Antunes O.A. Polikarpov I. J. Biol. Chem. 2000; 275: 20814-20821Google Scholar). Also the crystal structure of PI complexed with glucose has been elucidated with the correct amino acid sequence. 1T. A. Steitz, personal communication. Both of the isozymes share a similar α/β fold, and the polypeptide chain is distinctly folded into two domains of unequal size, the large and the small domain. These domains are separated by a large cleft forming the active site. The large domain consists of residues 19–76 and 212–457, whereas the small domain consists of residues 77–211 and 458–486. Comparison of the two crystal structures of hexokinase with and without bound glucose reveals that the conformational changes involved upon binding of the substrate are due to the domain movements. These conformational changes have earlier been observed spectroscopically for various substrates (16Feldman I. Kramp D.C. Biochemistry. 1978; 17: 1541-1547Google Scholar, 17Peters B.A. Neet K.E. J. Biol. Chem. 1978; 253: 6826-6831Google Scholar, 18Ohning G.V. Neet K.E. Biochemistry. 1983; 22: 2986-2995Google Scholar). In the era of proteomics, it is becoming increasingly important to understand the structure-function relationship of proteins using various biophysical tools (19Neet K.E. Lee J.C. Mol. Cell. Proteomics. 2002; 1: 415-420Google Scholar). Despite the importance of hexokinase as an enzyme, relatively few biophysical studies have been carried out on this protein. From the structures with the correct amino acid sequence available now, it is clear that the interface between the two domains is rich in acidic residues. Therefore, it will be of importance to study the effect of pH on domain interactions and hence the structure and of the enzyme. have yeast hexokinase A as a protein for studies and various biophysical techniques as transition 8-anilinonaphthalene-1-sulfonic CD, and fluorescence The results in conjunction with the structural data available have been in of interactions and their to the catalytic activity as well as the of the enzyme and the of at of pH. The results are to on the role of electrostatic interactions in the structure-function of the enzyme and have implications in understanding of the structural by the mutations to hexokinase A in these was obtained from as a of isozymes PI and The of isozymes was separated by using the of (9Womack F.C. Welch M.K. Nielsen J. Colowick S.P. Arch. Biochem. Biophys. 1973; 158: 451-457Google is on the of hexokinase isozymes to The PI was by pH The activity was using glucose and as substrates to The of obtained for the substrates was the of and the pH for the pH were from acid, and were from and were from or from was to the The pH of the was on a pH by acid or of and were for the pH-dependent or for pH and for pH and for pH for pH for pH and and pH and for studies on the of hexokinase A carried out by The protein were with the to their in obtained from The protein and the were for to into the The for the calorimetric studies was an or a from The were with the by the A of was and the data were an data to a in the of or in the of using The data were using the in the and were on a The was with In an of were and the data were as or in of the were obtained at an of with a of 50 at was at the of the and were in the and The protein for the and the were and were carried out at on a with a at a of using a A of 50 kDa and a number of residues for yeast hexokinase A crystal structure only were in the as well as fluorescence studies were carried out to the as well as to the transition were carried out on a with a and a were to the of the A protein of for the fluorescence and for the fluorescence was an of or was with an of and an of The was from to an of was and the was from to with of for and for of the and the transition were at were carried out at various pH to the for binding at a protein of of 50 was for the as a function of pH. The of was in a using an of at of New Scholar). Structural analysis was carried out on a using the II structures of hexokinase the and (10Kuser P.R. Krauchenco S. Antunes O.A. Polikarpov I. J. Biol. Chem. 2000; 275: 20814-20821Google Scholar) were The for the of is a of the Lee and B. J. Mol. Biol. 1971; Scholar) by and A. J. Mol. Biol. 1973; Scholar) in a of was The between the acidic residues were by the of the side chains of a acidic as the of yeast hexokinase A by in the pH of results in two independent thermal whereas only a single transition has been observed in the acidic pH of profiles of the protein by at with the of transitions observed by at acidic and alkaline pH that show and respectively A similar of observed for the of hexokinase both at the acidic and the alkaline pH suggests that the of is similar in both of the was observed to be between pH 8.5 and whereas pH it was independent transitions for thermal of yeast hexokinase A at pH and 8.5 have been Biochemistry. Scholar, F. A. G. G. J. Biochem. Scholar). The two transitions have been to from the of the two but these transitions have not been to the domains by a in the fluorescence at at pH 8.5 reveals only a single transition that to the first transition on the with the transition observed by and at hexokinase A of are in the hydrophobic in the smaller domain, whereas the larger domain only a single a of the of hexokinase A at pH 8.5 by the different techniques suggests that the first transition is to be due to the of the smaller domain, whereas the second transition corresponds to the of the larger for hexokinase A at different pH by at and fluorescence at at The the pH of as pH pH pH pH pH acidic thermal transitions of a single the merger of the two pH and the not is to suggesting a with both the domains between pH and the is less than due to the of of the as suggested by data The have been observed to at the of pH in whereas to the smaller domain, with to the larger domain in the alkaline pH suggesting that the interactions are the smaller domain to a than the larger domain. is in with the of J.F. Biochemistry. suggests that in the interactions the domain with a to a pH not any suggesting that the pH state is an acid-denatured state. pH the and structure of hexokinase it is important to the any to the structure and at any pH. and at in the pH and not any effect on as by and structure as by and fluorescence for the protein at a pH using the various of the transition of hexokinase A on the pH of the the of the first two transitions are observed alkaline or the single transition acidic of the second transition at alkaline In to the of electrostatic interactions to domain thermal at pH was carried out in the of pH was since was observed in the of at this pH with pH in the acidic pH range, a single transition has been pH the a single transition with a of in the of whereas the of the leads to a the transition observed was the and the transition could be into two The that the first transition is more than the second changes are not on not with no has been observed in a the of at whereas was at B. E. Sci. Scholar). The effect is at 100 and a to has the effect strong and calorimetric signal at pH us from out studies on the of the state of hexokinase A reveals at and and a one at In acidic the at these to changes in the pH. is a in the as a of in the pH at and the of the at with a in the a at pH by a this pH The at could be to since in this The changes observed at be to a in the as a of the loss of tertiary interactions. In alkaline the of hexokinase is to pH whereas at pH the protein of its tertiary interactions. is a in the at pH could be due to the of residues or due to the of in the alkaline pH the the of the hexokinase A show at and hexokinase A to a α/β of proteins (10Kuser P.R. Krauchenco S. Antunes O.A. Polikarpov I. J. Biol. Chem. 2000; 275: 20814-20821Google and the secondary structure content from the crystal structure consists of and The as a function of pH using the of Y.H. Biochemistry. is on data at are in I. In the pH content is is in with that from the crystal structure content of hexokinase A as a function of pH from the of of in a The secondary structure is to pH in the acidic at pH the protein with no observed at in pH is a in the suggesting that the pH state is an acid-denatured state. In the alkaline hexokinase A secondary structure at pH and a at pH with the acid-denatured state, the alkali-denatured state is more and it In both the acidic and the alkaline pH range, the show an at suggesting that the acid and is a A has residues the protein and residues, of are in the small domain and one in the large domain. The of from to was upon at to the changes in the of the residues or upon at in the both from and residues as well as due to the of transfer from to residues. the of the the protein with fluorescence from the residues. of the state have an of suggesting that the residues are in a The of the acid-denatured protein is to and that of the alkali-denatured protein is to that the of in the acid and the alkali-denatured protein to These data are in with the more secondary structure content for the acid-denatured protein as with the alkali-denatured protein. The of the protein upon from the state due to the of has been shown to to hydrophobic of proteins that become to G.V. Scholar). The results of binding to hexokinase A are shown in of the protein with as a function of pH from to and at pH suggests binding of in the acidic whereas no binding was observed at pH binding was at of binding in the acidic to an in the as well as a in the the of hydrophobic These studies the data at pH that show loss of tertiary interactions without any secondary structure The of binding of to the alkali-denatured protein suggests complete as by and by of at pH in to the conformational changes responsible for the effect of electrostatic interactions at alkaline pH between and the protein could from any binding pH of a and it is that yeast hexokinase A in two and pH Hexokinase A crystal structure reveals two and its calorimetric at pH 8.5 consists of two transitions that have been to the independent of the two domains Biochemistry. Scholar). acidic pH the thermal transitions of a single with of is similar to the results obtained at pH 8.5 in the of the thermal transition consists of a single with a to Biochemistry. Scholar). suggests that at the two domains could be with as in the of glucose at pH The pH for the activity of hexokinase is and it its activity pH with no activity obtained at pH A. G. Biochim. Biophys. Acta. Scholar). These results well with the domain merger as a function of pH observed by pH only one transition has been the enzyme has of its activity with that at pH two transitions are pH the transitions are and well with the first transition could be to the loss in the activity at pH using and CD, not any conformational changes between pH 8.5 and pH in the of the of the residues, M. J. J. Biochem. 1973; Scholar) that residues have no role in binding to the substrate or in and Biochemistry. 1978; 17: Scholar) have the pH of the enzyme and to the of the enzyme From the pH profiles of and for glucose observed of at acid residues that a loss of activity pH The were in the of and on for and the ionization was than for of the acid the high ionization to conformational changes with the and the observed for residues to their in the cleft with to the of the available crystal structures with the correct amino acid sequence of hexokinase B in the of glucose (10Kuser P.R. Krauchenco S. Antunes O.A. Polikarpov I. J. Biol. Chem. 2000; 275: 20814-20821Google Scholar) and hexokinase A in the of reveals the of acidic residues in the of the active site cleft that are different these residues, and are to the residues, and respectively in the the by to the substrate glucose II for between these residues and These residues are in the small domain and from the acidic residues in the or structure of and show in the in the suggesting that of these residues be in the as the of the residues be S. J. J. Mol. Biol. Scholar). The interactions with interactions from the active site by glucose be interactions between the two domains and resulting in the single transition observed by at pH 8.5 in the of glucose as by Biochemistry. Scholar). In the the two acidic residues, and are to than in the and be resulting in interactions. the overall by the of these acidic residues to one in the could be responsible for the of the resulting in two transitions observed by in the alkaline pH of or side chains the of the of the of the acidic residues in the active site of residues are at the active site cleft between the two of the acidic residues in the in both the and were using a of These residues are at the active site cleft between the two of the acidic residues in the in both the and were using a of in a M. Biochemistry. Scholar) have the domain in hexokinase to be of in and a and A The a and A in and of two The acidic residues are and suggests that the of these residues could be the domain movements. In the of results obtained by and Biochemistry. 1978; 17: Scholar) and the for domain suggested by M. Biochemistry. one of the for a single in the calorimetric profiles at acidic pH could be due to the of these acidic residues, the between the to the merger of the two a merger be responsible for the single transition observed at pH by calorimetry, well with the of and Biochemistry. 1978; 17: Scholar) of the in the of for acids in hexokinase active site. The binding of glucose to hexokinase has been to be with an and the is The relatively upon binding of glucose has been to the effect of of and Biochemistry. Scholar). has been that hexokinase large upon binding of a of to the of the conformational changes P. G. P. Biochemistry. Scholar, Lett. 2003; Scholar). The upon the of is and the has been to be due to the hydrophobic that the active of hexokinase form in the of but that an the W.S.J. Steitz T.A. Sci. S. A. 1978; Scholar). In the present the of at pH due to the of acidic residues at the of the cleft the with an due to the hydrophobic as by and Steitz W.S.J. Steitz T.A. Sci. S. A. 1978; Scholar). that the interactions in hexokinase are by a of hydrophobic and interactions. the hydrophobic interactions the the ionization of the acidic residues at the active site could as a between the and to domain movements for the catalytic activity of the enzyme. Biochemistry. Scholar) have observed that in at pH the of the high domain of hexokinase A the of the domain to to a single is to that of the glucose binding In the present at pH the less transition was to a larger than the transition with of suggests that the has a effect for one of the two domains by of the interactions. are known to the of amino acids that with the observed in a folded protein the J. M.K. J. Mol. Biol. Scholar, T. T. J. Biochem. Scholar, Scholar). could be that the of both the ionization of the acidic residues and leads to the of the the interactions. calorimetric profiles show a single transition at pH with a to at pH and suggesting the of the two domains in a was observed in the that the protein is in the state at these pH at pH the a of the at with a whereas is no in the The of the tertiary interactions be the hydrophobic is by binding fluorescence results at pH show a in the at without any in the suggesting the of the fluorescence due to tertiary interactions. pH the no in the with pH state but a complete loss of the at and is with the loss of secondary structure as shown by the pH the protein of the tertiary and it no transition upon by not or at suggesting that the pH state is an acid state. suggests that the loss of the structure due to a in the pH from to only the of tertiary whereas a in the pH leads to the loss of both the tertiary and the secondary interactions. of hexokinase A in was and a from the state to a state, has high secondary structure content as shown by CD, and it has an of suggesting of to the binding as by fluorescence at is for this state, suggesting that it could be a molten globule-like state. of yeast hexokinase A is to the II proteins as and that from state to the molten state without any and the is by a in the at whereas the proteins complete tertiary interactions T. Biochemistry. Scholar). transition pH is to the of of alkali-denatured protein less with the acid-denatured state but more with the urea-denatured protein. The to for the alkali-denatured protein with for the acid-denatured state, suggesting that the alkali-denatured protein is more than the acid-denatured protein. These results that the alkali-denatured protein is similar to a form observed in proteins Chem. Scholar, Biochemistry. 2002; Scholar) in the that it is less than the molten form and more structured than the protein. no of fluorescence was observed during it suggests that domain in a without any to be a since the conformational changes by different well with with an of pH In the results of the studies carried out show that interactions in hexokinase are by the pH and to be by the acidic residues at the of the active the structure as well as the of the These studies have implications in understanding the catalytic of hexokinase in the that the ionization state of these acidic residues be to domain movements with substrate binding and the release of The acid-denatured state of yeast hexokinase A has similar to that of the molten state, whereas the alkali-denatured protein is less structured than the acid-denatured protein and a studies of these acidic residues and their role in interactions be in understanding the catalytic of hexokinase T. A. Steitz for the of the hexokinase A structure with the correct amino acid sequence. M. for the of the for thermal studies and for during the of the A. for the of the

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