Linker residues regulate the activity and stability of hexokinase 2, a promising anticancer target
2020; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1074/jbc.ra120.015293
ISSN1083-351X
AutoresJuliana C. Ferreira, Abdul-Rahman Khrbtli, Cameron Lee Shetler, Samman Mansoor, Liaqat Ali, Özge Şensoy, Wael M. Rabeh,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoHexokinase (HK) catalyzes the first step in glucose metabolism, making it an exciting target for the inhibition of tumor initiation and progression due to their elevated glucose metabolism. The upregulation of hexokinase-2 (HK2) in many cancers and its limited expression in normal tissues make it a particularly attractive target for the selective inhibition of cancer growth and the eradication of tumors with limited side effects. The design of such safe and effective anticancer therapeutics requires the development of HK2-specific inhibitors that will not interfere with other HK isozymes. As HK2 is unique among HKs in having a catalytically active N-terminal domain (NTD), we have focused our attention on this region. We previously found that NTD activity is affected by the size of the linker helix-α13 that connects the N- and C-terminal domains of HK2. Three nonactive site residues (D447, S449, and K451) at the beginning of the linker helix-α13 have been found to regulate the NTD activity of HK2. Mutation of these residues led to increased dynamics, as shown via hydrogen deuterium exchange analysis and molecular dynamic simulations. D447A contributed the most to the enhanced dynamics of the NTD, with reduced calorimetric enthalpy of HK2. Similar residues exist in the C-terminal domain (CTD) but are unnecessary for HK1 and HK2 activity. Thus, we postulate these residues serve as a regulatory site for HK2 and may provide new directions for the design of anticancer therapeutics that reduce the rate of glycolysis in cancer through specific inhibition of HK2. Hexokinase (HK) catalyzes the first step in glucose metabolism, making it an exciting target for the inhibition of tumor initiation and progression due to their elevated glucose metabolism. The upregulation of hexokinase-2 (HK2) in many cancers and its limited expression in normal tissues make it a particularly attractive target for the selective inhibition of cancer growth and the eradication of tumors with limited side effects. The design of such safe and effective anticancer therapeutics requires the development of HK2-specific inhibitors that will not interfere with other HK isozymes. As HK2 is unique among HKs in having a catalytically active N-terminal domain (NTD), we have focused our attention on this region. We previously found that NTD activity is affected by the size of the linker helix-α13 that connects the N- and C-terminal domains of HK2. Three nonactive site residues (D447, S449, and K451) at the beginning of the linker helix-α13 have been found to regulate the NTD activity of HK2. Mutation of these residues led to increased dynamics, as shown via hydrogen deuterium exchange analysis and molecular dynamic simulations. D447A contributed the most to the enhanced dynamics of the NTD, with reduced calorimetric enthalpy of HK2. Similar residues exist in the C-terminal domain (CTD) but are unnecessary for HK1 and HK2 activity. Thus, we postulate these residues serve as a regulatory site for HK2 and may provide new directions for the design of anticancer therapeutics that reduce the rate of glycolysis in cancer through specific inhibition of HK2. The rate of glucose metabolism is elevated in different types of cancer that primarily utilize aerobic glycolysis, a phenomenon known as the Warburg effect (1Vander Heiden M.G. Cantley L.C. Thompson C.B. Understanding the Warburg effect: the metabolic requirements of cell proliferation.Science. 2009; 324: 1029-1033Crossref PubMed Scopus (8407) Google Scholar, 2Warburg O. On the origin of cancer cells.Science. 1956; 123: 309-314Crossref PubMed Scopus (8363) Google Scholar). An enhanced glucose metabolic rate is required to meet the increased energy needs and metabolite demands required to support rapid tumor progression (3Pedersen P.L. Warburg, me and Hexokinase 2: multiple discoveries of key molecular events underlying one of cancers' most common phenotypes, the "Warburg Effect", i.e., elevated glycolysis in the presence of oxygen.J. Bioenerg. Biomembr. 2007; 39: 211-222Crossref PubMed Scopus (345) Google Scholar). HK, the first enzyme in glucose metabolism, catalyzes the irreversible rate-limiting phosphorylation of glucose to glucose-6-phosphate (G6P). In addition to glycolysis, the HK reaction contributes to different pathways, including the tricarboxylic acid cycle and pentose phosphate pathway, for the synthesis of nucleotides, lipids, and amino acids required for rapid tumor growth (3Pedersen P.L. Warburg, me and Hexokinase 2: multiple discoveries of key molecular events underlying one of cancers' most common phenotypes, the "Warburg Effect", i.e., elevated glycolysis in the presence of oxygen.J. Bioenerg. Biomembr. 2007; 39: 211-222Crossref PubMed Scopus (345) Google Scholar, 4Patra K.C. Hay N. The pentose phosphate pathway and cancer.Trends Biochem. Sci. 2014; 39: 347-354Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar, 5DeWaal D. Nogueira V. Terry A.R. Patra K.C. Jeon S.M. Guzman G. Au J. Long C.P. Antoniewicz M.R. Hay N. Hexokinase-2 depletion inhibits glycolysis and induces oxidative phosphorylation in hepatocellular carcinoma and sensitizes to metformin.Nat. Commun. 2018; 9: 446Crossref PubMed Scopus (128) Google Scholar). As an effective regulator of glucose metabolism, HK can be targeted for the inhibition of cancer growth and the development of anticancer therapeutics. Five human HK isozymes with structurally identical NTD and CTD have been identified, including the new hexokinase domain containing 1 (HKDC1); however, HK4, known as glucokinase, is half the size and contains only a single domain (6Ardehali H. Yano Y. Printz R.L. Koch S. Whitesell R.R. May J.M. Granner D.K. Functional organization of mammalian hexokinase II. Retention of catalytic and regulatory functions in both the NH2- and COOH-terminal halves.J. Biol. Chem. 1996; 271: 1849-1852Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 7Ardehali H. Printz R.L. Whitesell R.R. May J.M. Granner D.K. Functional interaction between the N- and C-terminal halves of human hexokinase II.J. Biol. Chem. 1999; 274: 15986-15989Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 8Wilson J.E. Isozymes of mammalian hexokinase: structure, subcellular localization and metabolic function.J. Exp. Biol. 2003; 206: 2049-2057Crossref PubMed Scopus (643) Google Scholar, 9Hayes M.G. Urbanek M. Hivert M.F. Armstrong L.L. Morrison J. Guo C. Lowe L.P. Scheftner D.A. Pluzhnikov A. Levine D.M. McHugh C.P. Ackerman C.M. Bouchard L. Brisson D. Layden B.T. et al.Identification of HKDC1 and BACE2 as genes influencing glycemic traits during pregnancy through genome-wide association studies.Diabetes. 2013; 62: 3282-3291Crossref PubMed Scopus (64) Google Scholar). The conserved conformational fold of the NTD and CTD is constructed by small and large subdomains (Fig. 1, A and B) (6Ardehali H. Yano Y. Printz R.L. Koch S. Whitesell R.R. May J.M. Granner D.K. Functional organization of mammalian hexokinase II. Retention of catalytic and regulatory functions in both the NH2- and COOH-terminal halves.J. Biol. Chem. 1996; 271: 1849-1852Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 7Ardehali H. Printz R.L. Whitesell R.R. May J.M. Granner D.K. Functional interaction between the N- and C-terminal halves of human hexokinase II.J. Biol. Chem. 1999; 274: 15986-15989Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 10Aleshin A.E. Zeng C. Bartunik H.D. Fromm H.J. Honzatko R.B. Regulation of hexokinase I: crystal structure of recombinant human brain hexokinase complexed with glucose and phosphate.J. Mol. Biol. 1998; 282: 345-357Crossref PubMed Scopus (75) Google Scholar, 11Mulichak A.M. Wilson J.E. Padmanabhan K. Garavito R.M. The structure of mammalian hexokinase-1.Nat. Struct. Biol. 1998; 5: 555-560Crossref PubMed Scopus (90) Google Scholar, 12Nawaz M.H. Ferreira J.C. Nedyalkova L. Zhu H. Carrasco-Lopez C. Kirmizialtin S. Rabeh W.M. The catalytic inactivation of the N-half of human hexokinase 2 and structural and biochemical characterization of its mitochondrial conformation.Biosci. Rep. 2018; 38BSR20171666Crossref PubMed Scopus (14) Google Scholar). Each subdomain contains a β-sheet, where the active site is enclosed in a cleft between the β-sheets of the small and large subdomains. D209 and D657 are the catalytic residues positioned at the beginning of the catalytic helix-α5 and -α18 of the NTD and CTD, respectively. The linker helix-α13 is the last secondary structure of the NTD and protrudes from its active site to connect it to the CTD (Fig. 1D). The catalytic residues D209 and D657 are required to deprotonate the hydroxyl on C6 of glucose in preparation for its nucleophilic attack on the γ-phosphate of ATP (7Ardehali H. Printz R.L. Whitesell R.R. May J.M. Granner D.K. Functional interaction between the N- and C-terminal halves of human hexokinase II.J. Biol. Chem. 1999; 274: 15986-15989Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 12Nawaz M.H. Ferreira J.C. Nedyalkova L. Zhu H. Carrasco-Lopez C. Kirmizialtin S. Rabeh W.M. The catalytic inactivation of the N-half of human hexokinase 2 and structural and biochemical characterization of its mitochondrial conformation.Biosci. Rep. 2018; 38BSR20171666Crossref PubMed Scopus (14) Google Scholar, 13Aleshin A.E. Zeng C.B. Bourenkov G.P. Bartunik H.D. Fromm H.J. Honzatko R.B. The mechanism of regulation of hexokinase: new insights from the crystal structure of recombinant human brain hexokinase complexed with glucose and glucose-6-phosphate.Structure. 1998; 6: 39-50Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 14Ahn K.J. Kim J. Yun M. Park J.H. Lee J.D. Enzymatic properties of the N- and C-terminal halves of human hexokinase II.BMB Rep. 2009; 42: 350-355Crossref PubMed Scopus (24) Google Scholar). HK1 is ubiquitously expressed in all mammalian adult tissues and is the main housekeeping isozyme (8Wilson J.E. Isozymes of mammalian hexokinase: structure, subcellular localization and metabolic function.J. Exp. Biol. 2003; 206: 2049-2057Crossref PubMed Scopus (643) Google Scholar). On the other hand, HK2 is upregulated and predominantly expressed in various types of cancers, where it is required to enhance glycolysis for tumor growth and metastasis (5DeWaal D. Nogueira V. Terry A.R. Patra K.C. Jeon S.M. Guzman G. Au J. Long C.P. Antoniewicz M.R. Hay N. Hexokinase-2 depletion inhibits glycolysis and induces oxidative phosphorylation in hepatocellular carcinoma and sensitizes to metformin.Nat. Commun. 2018; 9: 446Crossref PubMed Scopus (128) Google Scholar, 15Gatenby R.A. Gillies R.J. Why do cancers have high aerobic glycolysis?.Nat. Rev. Cancer. 2004; 4: 891-899Crossref PubMed Scopus (3300) Google Scholar, 16Mathupala S.P. Ko Y.H. Pedersen P.L. Hexokinase-2 bound to mitochondria: cancer's stygian link to the "Warburg Effect" and a pivotal target for effective therapy.Semin. Cancer Biol. 2009; 19: 17-24Crossref PubMed Scopus (368) Google Scholar, 17Patra K.C. Wang Q. Bhaskar P.T. Miller L. Wang Z. Wheaton W. Chandel N. Laakso M. Muller W.J. Allen E.L. Jha A.K. Smolen G.A. Clasquin M.F. Robey R.B. Hay N. Hexokinase 2 is required for tumor initiation and maintenance and its systemic deletion is therapeutic in mouse models of cancer.Cancer Cell. 2013; 24: 213-228Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar, 18Anderson M. Marayati R. Moffitt R. Yeh J.J. Hexokinase 2 promotes tumor growth and metastasis by regulating lactate production in pancreatic cancer.Oncotarget. 2017; 8: 56081-56094Crossref PubMed Scopus (82) Google Scholar). Silencing HK2 in human hepatocellular carcinoma cells inhibited tumorigenesis and increased apoptosis (5DeWaal D. Nogueira V. Terry A.R. Patra K.C. Jeon S.M. Guzman G. Au J. Long C.P. Antoniewicz M.R. Hay N. Hexokinase-2 depletion inhibits glycolysis and induces oxidative phosphorylation in hepatocellular carcinoma and sensitizes to metformin.Nat. Commun. 2018; 9: 446Crossref PubMed Scopus (128) Google Scholar). HK2 was also found to be required for tumor initiation and maintenance in mouse models of lung and breast cancer (17Patra K.C. Wang Q. Bhaskar P.T. Miller L. Wang Z. Wheaton W. Chandel N. Laakso M. Muller W.J. Allen E.L. Jha A.K. Smolen G.A. Clasquin M.F. Robey R.B. Hay N. Hexokinase 2 is required for tumor initiation and maintenance and its systemic deletion is therapeutic in mouse models of cancer.Cancer Cell. 2013; 24: 213-228Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). In addition, HK2 binding to the outer mitochondrial membrane and its interaction with the voltage-dependent anion channel (VDAC) suppress apoptosis and enhance tumor cell survival (19Pastorino J.G. Hoek J.B. Hexokinase II: the integration of energy metabolism and control of apoptosis.Curr. Med. Chem. 2003; 10: 1535-1551Crossref PubMed Scopus (199) Google Scholar, 20Pastorino J.G. Shulga N. Hoek J.B. Mitochondrial binding of hexokinase II inhibits Bax-induced cytochrome c release and apoptosis.J. Biol. Chem. 2002; 277: 7610-7618Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar, 21Shulga N. Pastorino J.G. Hexokinase II binding to mitochondria is necessary for Kupffer cell activation and is potentiated by ethanol exposure.J. Biol. Chem. 2014; 289: 26213-26225Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). Though minimally expressed in normal tissues, HK2 is found in embryonic tissues and selectively expressed at the basal level in adipose and muscle adult tissues (5DeWaal D. Nogueira V. Terry A.R. Patra K.C. Jeon S.M. Guzman G. Au J. Long C.P. Antoniewicz M.R. Hay N. Hexokinase-2 depletion inhibits glycolysis and induces oxidative phosphorylation in hepatocellular carcinoma and sensitizes to metformin.Nat. Commun. 2018; 9: 446Crossref PubMed Scopus (128) Google Scholar, 15Gatenby R.A. Gillies R.J. Why do cancers have high aerobic glycolysis?.Nat. Rev. Cancer. 2004; 4: 891-899Crossref PubMed Scopus (3300) Google Scholar). The biological importance of HK2 for the survival, progression, and chemoresistance of a variety of tumor types and its low abundance in normal tissues make it an attractive target for the development of anticancer therapeutics (17Patra K.C. Wang Q. Bhaskar P.T. Miller L. Wang Z. Wheaton W. Chandel N. Laakso M. Muller W.J. Allen E.L. Jha A.K. Smolen G.A. Clasquin M.F. Robey R.B. Hay N. Hexokinase 2 is required for tumor initiation and maintenance and its systemic deletion is therapeutic in mouse models of cancer.Cancer Cell. 2013; 24: 213-228Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar, 18Anderson M. Marayati R. Moffitt R. Yeh J.J. Hexokinase 2 promotes tumor growth and metastasis by regulating lactate production in pancreatic cancer.Oncotarget. 2017; 8: 56081-56094Crossref PubMed Scopus (82) Google Scholar, 22Pedersen P.L. Mathupala S. Rempel A. Geschwind J.F. Ko Y.H. Mitochondrial bound type II hexokinase: a key player in the growth and survival of many cancers and an ideal prospect for therapeutic intervention.Biochim. Biophys. Acta. 2002; 1555: 14-20Crossref PubMed Scopus (286) Google Scholar, 23Cheong H. Lu C. Lindsten T. Thompson C.B. Therapeutic targets in cancer cell metabolism and autophagy.Nat. Biotechnol. 2012; 30: 671-678Crossref PubMed Scopus (250) Google Scholar, 24Jang M. Kim S.S. Lee J. Cancer cell metabolism: implications for therapeutic targets.Exp. Mol. Med. 2013; 45: e45Crossref PubMed Scopus (192) Google Scholar, 25Suh D.H. Kim M.A. Kim H. Kim M.K. Kim H.S. Chung H.H. Kim Y.B. Song Y.S. Association of overexpression of hexokinase II with chemoresistance in epithelial ovarian cancer.Clin. Exp. Med. 2014; 14: 345-353Crossref PubMed Scopus (53) Google Scholar, 26Li X.B. Gu J.D. Zhou Q.H. Review of aerobic glycolysis and its key enzymes - new targets for lung cancer therapy.Thorac. Cancer. 2015; 6: 17-24Crossref PubMed Scopus (134) Google Scholar, 27Garcia S.N. Guedes R.C. Marques M.M. Unlocking the potential of HK2 in cancer metabolism and therapeutics.Curr. Med. Chem. 2019; 26: 7285-7322Crossref PubMed Scopus (25) Google Scholar). Currently, the most commonly used cancer treatment is still chemotherapy, a procedure with high side effects due to its low specificity for cancer cells (27Garcia S.N. Guedes R.C. Marques M.M. Unlocking the potential of HK2 in cancer metabolism and therapeutics.Curr. Med. Chem. 2019; 26: 7285-7322Crossref PubMed Scopus (25) Google Scholar). However, inhibitors of glucose metabolism have been proposed as an effective therapeutic strategy for cancer treatment (5DeWaal D. Nogueira V. Terry A.R. Patra K.C. Jeon S.M. Guzman G. Au J. Long C.P. Antoniewicz M.R. Hay N. Hexokinase-2 depletion inhibits glycolysis and induces oxidative phosphorylation in hepatocellular carcinoma and sensitizes to metformin.Nat. Commun. 2018; 9: 446Crossref PubMed Scopus (128) Google Scholar, 27Garcia S.N. Guedes R.C. Marques M.M. Unlocking the potential of HK2 in cancer metabolism and therapeutics.Curr. Med. Chem. 2019; 26: 7285-7322Crossref PubMed Scopus (25) Google Scholar, 28Wang H. Wang L. Zhang Y. Wang J. Deng Y. Lin D. Inhibition of glycolytic enzyme hexokinase II (HK2) suppresses lung tumor growth.Cancer Cell Int. 2016; 16: 9Crossref PubMed Scopus (35) Google Scholar). Due to the highly conserved identity of the active site residues of human HKs, one of the greatest challenges in the design and development of anticancer inhibitors based on the HK reaction is the preferential targeting of HK2 over the other human isozymes (12Nawaz M.H. Ferreira J.C. Nedyalkova L. Zhu H. Carrasco-Lopez C. Kirmizialtin S. Rabeh W.M. The catalytic inactivation of the N-half of human hexokinase 2 and structural and biochemical characterization of its mitochondrial conformation.Biosci. Rep. 2018; 38BSR20171666Crossref PubMed Scopus (14) Google Scholar, 17Patra K.C. Wang Q. Bhaskar P.T. Miller L. Wang Z. Wheaton W. Chandel N. Laakso M. Muller W.J. Allen E.L. Jha A.K. Smolen G.A. Clasquin M.F. Robey R.B. Hay N. Hexokinase 2 is required for tumor initiation and maintenance and its systemic deletion is therapeutic in mouse models of cancer.Cancer Cell. 2013; 24: 213-228Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). Anticancer therapeutics that target cancer growth will have to specifically inhibit HK2 and avoid interactions with HK in normal tissues. For this reason, successful drug candidates need to bind outside the highly conserved HK active site to avoid the inhibition of all human isozymes. Although HK1 and HK2 share high structural fold similarity, they do exhibit biochemical differences that can be explored for the development of HK2-specific inhibitors. The CTD is catalytically active in all human HKs. Despite its structural fold similarity to the CTD, the NTD is inactive in all human isozymes except HK2. We previously found that the size of the linker helix-α13 regulates the activity of the NTD of HK2 (12Nawaz M.H. Ferreira J.C. Nedyalkova L. Zhu H. Carrasco-Lopez C. Kirmizialtin S. Rabeh W.M. The catalytic inactivation of the N-half of human hexokinase 2 and structural and biochemical characterization of its mitochondrial conformation.Biosci. Rep. 2018; 38BSR20171666Crossref PubMed Scopus (14) Google Scholar). When expressed separately from the full-length (FL) enzyme with four of the eight helical turns of the linker helix-α13, the NTD variant was inactive. However, increasing the number of helical turns of the linker helix-α13 recovered the activity of the NTD variant. The ability of the linker helix-α13 to regulate the NTD activity of HK2 makes it an excellent target for the development of specific inhibitors of HK2's active NTD without interacting with or binding to the HK active site. In this study, we investigated the roles of the linker helix-α13 residues on the catalytic activity of the NTD of HK2 to identify a possible regulatory site. Site-directed mutagenesis was used to determine the roles of residues in the linker helix-α13 and its network of interactions on the NTD activity of HK2. We identified three residues (D447, S449, and K451) at the beginning of the linker helix-α13 that regulate the activity of the NTD of HK2. These residues have been found to maintain the conformational stability around the NTD active site, enhancing the structural stability of the linker helix-α13 and its interaction with the catalytic helix-α5 of the NTD. CTD of HK1 and that of HK2 contain identical residues to those in the NTD regulatory site, but these residues were unable to control the activity of the CTD. The newly identified NTD regulatory site of HK2 is a promising target for the design of anticancer therapeutics that would reduce the rate of glycolysis in cancer through specific inhibition of the upregulated HK2. Human HK2, similar to isozymes HK1, HK3, and HKDC1, is a homodimer in which each monomer is split into two structurally identical domains, the NTD and CTD (Fig. 1, A–C) (10Aleshin A.E. Zeng C. Bartunik H.D. Fromm H.J. Honzatko R.B. Regulation of hexokinase I: crystal structure of recombinant human brain hexokinase complexed with glucose and phosphate.J. Mol. Biol. 1998; 282: 345-357Crossref PubMed Scopus (75) Google Scholar, 11Mulichak A.M. Wilson J.E. Padmanabhan K. Garavito R.M. The structure of mammalian hexokinase-1.Nat. Struct. Biol. 1998; 5: 555-560Crossref PubMed Scopus (90) Google Scholar, 12Nawaz M.H. Ferreira J.C. Nedyalkova L. Zhu H. Carrasco-Lopez C. Kirmizialtin S. Rabeh W.M. The catalytic inactivation of the N-half of human hexokinase 2 and structural and biochemical characterization of its mitochondrial conformation.Biosci. Rep. 2018; 38BSR20171666Crossref PubMed Scopus (14) Google Scholar). The overall α/β structural fold of the NTD and CTD is conserved among the mammalian HK family (10Aleshin A.E. Zeng C. Bartunik H.D. Fromm H.J. Honzatko R.B. Regulation of hexokinase I: crystal structure of recombinant human brain hexokinase complexed with glucose and phosphate.J. Mol. Biol. 1998; 282: 345-357Crossref PubMed Scopus (75) Google Scholar, 29Kamata K. Mitsuya M. Nishimura T. Eiki J. Nagata Y. Structural basis for allosteric regulation of the monomeric allosteric enzyme human glucokinase.Structure. 2004; 12: 429-438Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar, 30Kawai S. Mukai T. Mori S. Mikami B. Murata K. Hypothesis: structures, evolution, and ancestor of glucose kinases in the hexokinase family.J. Biosci. Bioeng. 2005; 99: 320-330Crossref PubMed Scopus (77) Google Scholar, 31Kuettner E.B. Kettner K. Keim A. Svergun D.I. Volke D. Singer D. Hoffmann R. Muller E.C. Otto A. Kriegel T.M. Strater N. Crystal structure of hexokinase KlHxk1 of Kluyveromyces lactis: a molecular basis for understanding the control of yeast hexokinase functions via covalent modification and oligomerization.J. Biol. Chem. 2010; 285: 41019-41033Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 32Kuser P.R. Krauchenco S. Antunes O.A. Polikarpov I. The high resolution crystal structure of yeast hexokinase PII with the correct primary sequence provides new insights into its mechanism of action.J. Biol. Chem. 2000; 275: 20814-20821Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 33Lin H. Zeng J. Xie R. Schulz M.J. Tedesco R. Qu J. Erhard K.F. Mack J.F. Raha K. Rendina A.R. Szewczuk L.M. Kratz P.M. Jurewicz A.J. Cecconie T. Martens S. et al.Discovery of a novel 2,6-disubstituted glucosamine series of potent and selective hexokinase 2 inhibitors.ACS Med. Chem. Lett. 2016; 7: 217-222Crossref PubMed Scopus (36) Google Scholar), and the active site is located in a cleft between the large and small subdomains. In mammalian HK, the glucose binding site is conserved and includes the catalytic residues D209 and D657 in the NTD and CTD, respectively (12Nawaz M.H. Ferreira J.C. Nedyalkova L. Zhu H. Carrasco-Lopez C. Kirmizialtin S. Rabeh W.M. The catalytic inactivation of the N-half of human hexokinase 2 and structural and biochemical characterization of its mitochondrial conformation.Biosci. Rep. 2018; 38BSR20171666Crossref PubMed Scopus (14) Google Scholar). We previously reported that the linker helix-α13 (residues G448–Q478) is important for maintaining the catalytic activity of the NTD (Fig. 1, A and B), which is composed of a long eight-turn α-helix (12Nawaz M.H. Ferreira J.C. Nedyalkova L. Zhu H. Carrasco-Lopez C. Kirmizialtin S. Rabeh W.M. The catalytic inactivation of the N-half of human hexokinase 2 and structural and biochemical characterization of its mitochondrial conformation.Biosci. Rep. 2018; 38BSR20171666Crossref PubMed Scopus (14) Google Scholar). The linker helix-α13 protrudes from the active site at the end of the NTD, thus connecting it to the CTD. It is also perpendicular to the catalytic helix-α5 that carries the catalytic residue D209 (Fig. 1D). In this study, we investigated the roles of residues of the linker helix-α13 on the catalytic activity of the NTD of HK2 to identify a possible regulatory site, which would be targeted in the design of anticancer therapeutics to reduce the rate of glycolysis in cancer through the inhibition of upregulated HK2. To assess the role of residues of the linker helix-α13 in the catalytic activity of the NTD of HK2, site-directed mutagenesis was used to introduce mutants into the linker helix-α13 and its network of interactions. Since both the NTD and CTD of HK2 are catalytically active, the enzymatic rate of the NTD was measured in the FL variant in the presence of D657A mutant to catalytically inactive the CTD. All HK2 mutants were expressed and purified using Ni-NTA affinity chromatography followed by size-exclusion chromatography as described before (12Nawaz M.H. Ferreira J.C. Nedyalkova L. Zhu H. Carrasco-Lopez C. Kirmizialtin S. Rabeh W.M. The catalytic inactivation of the N-half of human hexokinase 2 and structural and biochemical characterization of its mitochondrial conformation.Biosci. Rep. 2018; 38BSR20171666Crossref PubMed Scopus (14) Google Scholar). The protein purity was assessed using SDS-PAGE with high purity to enable detailed characterization of the WT and mutant enzymes (Fig. S1). The enzymatic rate of HK2 mutants was measured at saturated concentrations of glucose and ATP using the coupled G6P dehydrogenase assay. First, the interaction of the linker helix-α13 with the catalytic helix-α5 and loop444−447 of the large subdomain in the NTD was investigated. The last β-strand of the large subdomain extends outward as loop444−447 to connect to the linker helix-α13, where D447 of loop444−447 forms a salt bridge (3.1 Å) with K451 of the linker helix-α13 (Fig. 1E). K451 is conserved in human HKs, while D447 is partially conserved and replaced by S447 in HK1 and HKDC1 (Fig. S2). The enzyme titration assay showed that D447A and K451A mutants were surprisingly catalytically inactive, where the alanine substitution of each residue was introduced in the presence of D657A in the FL variant (Fig. 2, A and G). These alanine substitutions at nonactive site residues were able to eliminate the catalytic activity of NTD of HK2. To recover the activity of D447A and K451A mutants, alternative amino acid substitutions were introduced. The NTD activity was partially recovered in the presence of D447E, D447N, D447S, and K451R (Fig. 2, A and G). The amino acid substitutions D447E and K451R conserved the ionic charges but increased the size of the side chains for these residues, thus decreasing the distance between D447 and K451. The partial recovery of the NTD activity of D447E and K451R mutants indicates that not only the charge but also the distance between D447 and K451 is important in maintaining the activity of the NTD. Therefore, D447 and K451 facilitate strong interactions and mediate a specific distance between the linker helix-α13 and loop444−447 for optimum activity of the NTD of HK2. An intramolecular interaction is observed between E446 and R444 (3.1 and 4.1 Å) in loop444−447 (Fig. 1E), where the NTD enzymatic rate was also reduced by 57% ± 1% and 74% ± 1% in the presence of R444L and E446A mutations, respectively (Fig. 2, B and G). Interestingly, only HK2 harbors a positively charged residue R444, whereas the corresponding residue is leucine in HK1 and HKDC1, proline in HK3, and E440 in HK4 (Fig. S2). An alternative amino acid substitution, E446N, recovered the enzymatic activity of the NTD. In contrast to D447 and K451, the intramolecular interactions of loop444−447 reduced, but did not eliminate the catalytic activity of the NTD of HK2. Furthermore, E446 forms an additional bond with K418 of the large subdomain (4.4 Å), where both residues are conserved in the NTD and CTD of all human HK isozymes (Fig. 1E). The K418A mutant increased the activity of the NTD at 146% ± 1% compared with the control (Fig. 2, B and G). In the NTD of HK2, the catalytic helix-α5 (residues: D209–D220) is a small three-turn α-helix that harbors the catalytic residue D209. The catalytic helix-α5 is perpendicular to the linker helix-α13, and both are wedged between the two β-sheets of the large and small subdomains (Fig. 1B). The linker helix-α13 interacts with the catalytic helix-α5 at three sites. First, S449 at the beginning of the linker helix-α13 interacts with the side chain of T213 (3.0 Å) and peptide backbone of D209 (3.1 Å), both of which are located on the catalytic helix-α5 (Fig. 1D). Similar to D447A and K451A, the introduction of S449A eliminated the catalytic activity of the NTD of HK2 (Fig. 2, C and H). Multiple amino acid substitutions were introduced at S449 to evaluate its role in catalysis, where S449N mutant slightly recovered the NTD activity. Interestingly, S449T was inactive, although threonine has the same hydroxyl functional group on its side chain as serine, and S449D slightly increased the NTD activity (Fig. 2H). This indicates that not only the hydrogen bonding int
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