The enzyme hexokinase hk catalyzes the reaction between glucose and adenosine triphosphate atp to form glucose6phosphate g6p and adenosine diphosphate adp. Furthermore, mammalian sperm contain also atypical isozymes of phosphoglucose isomerase, triosephosphate isomerase, phosphoglycerate mutase, enolase and others. Hexokinase colorimetric assay kit mak091 technical bulletin. All of the hexokinases can mediate phosphorylation of glucose to glucose6phosphate g6p, which is the first step of both glycogen synthesis and glycolysis. Hexokinase in mammalian tissues exists as four isoenzymes with distinct kinetic properties and tissue distribution 1, 2. Deletion of hxk2, the gene which encodes hexokinase ii, dramatically changed the physiology of s. It was found that the fraction hk ii is present in every malignant tumor and in placenta tissue. Isozymes of hexokinase different metabolic roles hexokinase. Hexokinaseii positively regulates glucose starvationinduced.
Because the isozymes have different kinetic features, this has permitted them to become optimally adapted to the needs of the specific tissue in which they are the major isozyme. Determine whether the phrase described glucokinase. In most organisms, glucose is the most important substrate of hexokinases, and glucose6phosphate is the most important product. Hexokinase 1 hk1, hexokinase 2 hk2, and hexokinase 3 hk3 are allosterically inhibited by their product, glucose6phosphate, but hk4 is not sensitive to glucose6phosphate concentrations wilson, 1995. Multiple forms of human red blood cell hexokinase journal of. The mode of the inhibition by the thiol inhibitor was different for the type i. The catalytic properties of hexokinase i are dependent on product inhibition as well as on the action of phosphate. With fructose as the variable substate, michaelis constant values for the scutellum enzyme were about onethird those of the endosperm enzyme 0. Vmax can be reached in the presence of a competitive inhibitor. In the presence of nicotinamide adenine dinucleotide nad, g6p is oxidized by the enzyme glucose6phosphate dehydrogenase g6. The existence of isozymes permits the finetuning of metabolism to meet the particular needs of a.
Read expression, purification, and characterization of a recombinant erythroidspecific hexokinase isozyme, blood cells, molecules and diseases on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Glk1 polymerization inhibits its hexokinase activity, and the. Hexokinase iib constitutes the principal regulated isoform in many cell types and is increased in many cancers. After glucose binds to hexokinase, the two lobes close, bringing carbon 6 of glucose closer to the terminal phosphate of atp. Phosphoglycerate kinase transfers a phosphoryl group from 1,3biphosphoglycerate to adp to form atp twice bc since the aldolase reaction were working in double substrates. In mammals four hexokinase isozymes have been identified named iiv, which differ in their catalytic. Hexokinase 1, phosphofructokinase m, and glutathione stransferase mu class 5 1. Hexokinase isozyme patterns of human uterine carcinomas and myomas were studied using cellulose acetate membrane electrophoresis.
It is well known that glycolysis is regulated by three regulator enzymes hexokinase hk, phosphofructokinase pfk and pyruvate kinase pk. These multiple forms can be detected by electrophoresis of crude tissue extracts followed by specific staining for hexokinase activity 8, 9. The hxk2 null mutant strain displayed fully oxidative growth at high glucose concentrations in early exponential batch cultures, resulting in an initial absence of. Uterine myomas contained only type i hexokinase, like normal myometrium, and had similar specific activity to the latter. Mar 01, 2010 molecular complex of three testisspecific isozymes associated with the mouse sperm fibrous sheath. Keith tornheim, in encyclopedia of endocrine diseases second edition, 2018. Hexokinase activity is determined by a coupled enzyme assay, in which glucose is converted to glucose6phosphate by hexokinase, which is oxidized by glucose6phosphate dehydrogenase to form nadh.
Physiological properties of saccharomyces cerevisiae from. Mammals show four hexokinase isoforms type iiv grossbard and schimke, 1966. The abnormality in the pattern of hexokinase isozymes in human tumors and the. Identification of the cdna for human red blood cellspecific. However, they are expressed in different tissues and have different kinetic and regulatory properties.
What is the difference between hexokinase and glucokinase. Expression, purification, and characterization of a. Glucokinase is a liver enzyme, whereas hexokinase is. This movement also excludes water, which could otherwise hydrolyze atp. Although they have the same catalytic activity, they are physically distinct and differ in electrophoretic mobility and liability to inhibitors. Enzymes with very broad substrate specificities were excluded from the initial definition of the term.
Multiple glycolytic enzymes are tightly bound to the fibrous sheath of mouse spermatozoa. The multiple forms of mammalian hexokinase and their significance. Isoenzymes isozymes are multiple forms of the enzyme that have the same catalytic activity. Multiple active sites that can exist on multiple subunits. In mammals there are four hexokinase isozymes with different tissue distributions. Uterine carcinomas of the cervix and corpus contained type ii hexokinase as well as type i, unlike normal uterine cervical epithelium and endometrium which contained only type i. The type i and type ii isozymes of hexokinase coexist in insulinsensitive tissues, such as cardiac and skeletal muscle and adipose tissue. Effects of free magnesium and alkali ions on the conformation and glucosebinding strength of yeast hexokinse isozymes. Substantial roles of hexokinase and fructokinase in the. This appeared to represent the cdna for the red blood cell rbcspecific hk isozyme hk r described in our previous study murakami et al. Such a method has been described as the most efficient for the study of the hk isozymic pattern in mammalian red blood cells 8, 9 and has been here proved to be adequate to easily separate isozymes of. Distinguishing the type i and type ii isozymes of hexokinase. Hexokinase, phosphofructokinase and pyruvate kinase isozyrnes.
Multiple hexokinases of rat tissues journal of biological chemistry. Hexokinase colorimetric assay kit mak091 technical. The hexokinase activities of carcinoma tissues were also higher than those of control tissues. A read is counted each time someone views a publication summary such as the title, abstract, and list of authors, clicks on a figure, or views or downloads the fulltext. Isozymes of hexokinase different metabolic roles hexokinase muscle k 05 01 mm from bch 110 at university of california, riverside. Isozymes also known as isoenzymes or more generally as multiple forms of enzymes are enzymes that differ in amino acid sequence but catalyze the same chemical reaction. Sonication was used in the presence of kcltriton x100 to convert hexokinase activity in the. Oct 17, 2014 hexokinase ii phosphorylates glucose to regulate glucose metabolism but also functions as a protective signaling molecule. Multiple forms of hexokinasehexokinase in mammalian liver exists as four isoenzymes. These enzymes catalyze the same reaction converting glucose to glucose6phosphate. The enzyme hk is known to consist of three isozymes a, b, and c separable from each other and from gk by starch gel.
The structure and mechanism of hexokinase proteopedia, life. Importantly, glucose 6phosphate is an inhibitor of hexokinase, so if the other. Hexokinase isozyme distribution in human skeletal muscle diabetes. Both of these enzymes are iso enzymes, which means that both of them have the same chemical natures and have same action but have different physical properties and site of action etc. In this context, despite the multiple functions displayed by atp in hexokinase activity, it is. There are 117 instances of this domain found in the pdb. Multiple hexokinase isozymes have been found in most vertebrates. Isozyme distribution of hexokinase, phosphofructokinase. Glucokinase hexokinase hexokinase 4 hexokinase 1 livers enzyme it is a rbcs enzyme found in liver found in multiple tissues but we will focus on rbcs but what is the purpose for this distribution glucose6phosphate may then be metabolized in glycolysis, which generates energy in. Evidently, in some packing environments and even in the presence of glucose and. It is clear that these are distinct in character, and responsive to quite different transcription factors.
The mode of the inhibition by the thiol inhibitor was different for the type i isoenzymes obtained from different tissues, suggesting that the type i isoenzymes from different tissues were not identical proteins. The first step of glycolysis phosphorylates glucose. Hexokinase isozyme distribution in human skeletal muscle. In part a of these experiments, magnesium was maintained at 10 mm in excess of the adp and atp. The proportion of these isozymic forms appears to be adaptive and can be alterated by developmental, hormonal, and dietary. Hexokinase is the first key enzyme in glycolysis because it traps glucose within cells. Isozyme distribution of hexokinase, phosphofructokinase and. Identification of the cdna for human red blood cell. Abstract hexokinase i is comprised of homologous n and cterminal domains, and binds to the outer membrane of mitochondria. Enzyme kinetics is the study of chemical reactions catalyzed by enzymes.
Recently, we reported on hexokinase isozyme distribution and regulatory proper ties in lymphoid cells 181. The decrease of glycolytic enzyme hexokinase 1 accelerates. Hexokinase iv blue curve in the plot below, also called glucokinase, is the predominant isozyme in liver and has distinct kinetic properties from the other hexokinase. Hexokinase might also be considered part of the glucose transport system because it maintains a high transmembrane gradient for free glucose. Discrimination of antigenic site and thiolinhibitor. The presence of multiple hexokinase isozymes iiv for the phosphorylation of glucose enables specific organs to regulate carbohydrate metabolism in a unique way. Hexokinase ii integrates energy metabolism and cellular. Spermatogenic cellspecific type 1 hexokinase is the. Hexokinase ia is found in all mammalian tissues, and is considered a housekeeping enzyme, unaffected by most physiological, hormonal, and metabolic changes.
Jan 23, 2014 hexokinase ii hkii catalyzes the first step of glycolysis. Adp and glucose6p for the three hexokinase isoenzymes, it is. Hexokinase is nominally the first step in glycolysis, but it is not the major point of regulation because glucose 6phosphate is a branch point leading to glycogen and to the pentose phosphate pathway. In adult drosophila homogenates, three main hexokinase bands were observed by starch gel electrophoresis 9. All the cells contained pk type k, and the hybrid k,m. Molecular complex of three testisspecific isozymes. Structural comparisons of three vertebrate hexokinases having a molecular weight of. This message will disappear when all data is loaded. Difference in kinetic properties between hexokinase type i isoenzymes from various rat tissues with reference to the effect of a thiol inhibitor. These enzymes usually display different kinetic parameters e. The hexokinase types iiii all have a high affinity for glucose and become subject to inhibition in the presence of glucose6phosphate the first step reaction product in glycolysis. Studies of hexokinase isozymes in human skeletal muscle are limited. Evidence for the presence of a cysteine residue in the. Hexokinase 1, phosphofructokinase m, and glutathione stransferase mu class 5.
In vivo, a large fraction of hexokinase i is bound to the mitochondrial outer membrane, where the enzyme adopts a tetrameric assembly. Phosphoglycerate kinase, aka the breakeven reaction 2 atp are produced. Note that there may be multiple copies of the domain in a single pdb structure, since many structures contain multiple copies of the same protein sequence. At least two hexokinase isozymes are apparent, both staining very. In mammals, four hexokinase isozymes have been characterized, including hk1, hk2, and hk3 with two domains 100 kda that are structurally similar to the single domain hk4 52 kda, also known as glucokinase. A comparative study by a single chromatographic procedure of. For rat skeletal muscle, the predominant type is hexokinase ii 1,4,5. Iso means the same and isoenzyme means the same enzyme. The expression and activities of glucokinase gk and hexokinase hk were assessed in different tissues of rainbow trout oncorhynchus mykiss under different feeding conditions fed, fasted for 14 days, and refed for 7 days.
Hexokinase vs glucukinase another example is the enzymes hexokinase and glucokinase hexokinase iv. Structure, subcellular localization and metabolic function. The hexokinase activities of carcinoma tissues were also higher than those of control. Like other catalysts, enzymes provide an alternate pathway from substrate to product with a lower ea yet the enzyme does not alter the equilibrium between substrates and products. Apart from acting as a hexokinase, multiple moonlighting roles have been assigned to hexokinases.
Multiple glucosesensing mechanisms are present in yeast. Hexokinase, phosphofructokinase and pyruvate kinase isozyrnes in lymphocyte subpopulations. Glucokinase and hexokinase expression and activities in. Stressgoverned expression and purification of human type ii. A hexokinase is an enzyme that phosphorylates hexoses sixcarbon sugars, forming hexose phosphate. Two different hki cdnas were identified with different tissue distributions. Evidence for the existence in human erythrocytes of.
Difference in kinetic properties between hexokinase type i. The yeast hexokinase glk1 is an actinfold protein that forms polymers in response to binding its substrates and products. Hexokinase isoenzyme type i was purified from various rat tissues, and was subjected to kinetic studies in the presence or the absence of pchloromercuribenzenesulphonate. A comparative study by a single chromatographic procedure. Three encode isozymes hk1, hk2 and hk3 of 100 kda that contain two homologous sequences hk domain. Isozymes have similar or identical substrate specificities.
Hexokinaseii positively regulates glucose starvation. The hexokinase hxk sugarsensing pathway, the trehalose6phosphate t6p pathway, and target of the rapamycin tor kinase pathway all play roles in the presence of excess sugar, whereas snfrelated protein kinases snrks and the cs1 bzip transcription factor network play important roles when sugar is in limited supply smeekens et al. The term isozyme was coined by markert and moller 1959 to refer to the occurrence of multiple forms of a given enzyme within the same organism. Hk has high affinity for glucose the k m value is below 0. In mammals four hexokinase isozymes have been identified named iiv, which differ in their catalytic and regulatory properties as well as in. Hexokinase type i is the predominant form in the muscle while hexokinase type ii is the predominant form in myocytes. Hexokinase iv blue curve in the plot below, also called glucokinase, is the predominant isozyme in liver and has distinct kinetic properties from the other. Hexokinase ii phosphorylates glucose to regulate glucose metabolism but also functions as a protective signaling molecule.
The hexokinase colorimetric assay kit provides a simple and direct procedure for measuring hexokinase activity in a v ariety of samples. Thus the existence of multiple genes encoding the isozymes of hexokinase does offer the flexibility in. Rat brain hk1 was shown to have autophosphorylating and protein kinase activity adams et al. Four distinct isozymes of hexokinase were separated by ion exchange chromatography gonzalez et al. Multiple forms of hexokinases are known to exist in higher organisms 17. Hexokinase phosphorylates hexoses transfer an inorganic phosphate group from atp to a hexose and form hexose phosphate. Hexokinase, phosphofructokinase and pyruvate kinase. Based on an early report that the purified type i isozyme was stable at 45 degrees c whereas the purified type ii isozyme was not, investigators in a number of studies have used heat lability as a criterion for distinguishing these isozymes in crude tissue.
Hexokinase possesses the ability to transfer an inorganic phosphate group from atp to a substrate. The distribution of electrophoretic fractions of hexokinase hk e. Walkeron the presence of two soluble glucosephosphorylating enzymes in adult liver and the. To investigate whether attenuated glycolytic activity modulates tumor progression, the effects of silencing the first and ratelimiting glycolytic enzyme hexokinase hk isozymes hk1 and hk2 were examined. The glycolytic enzyme hexokinase hkii participates in tumor glycolysis and the progression of various cancers, but its clinicopathological effect on the progression of tongue squamous cell carcinoma tscc and its role in glycolysis, autophagy, and the epithelialmesenchymal transition of tscc in a hypoxic microenvironment remain unknown.
A common feature of these isozymes, also present in the ancestral hexokinase, is the. Hexokinase iv blue curve in the plot below, also called glucokinase, is the predominant isozyme in liver and has distinct kinetic properties from the other hexokinase isozymes. Hexokinase isozyme patterns of human uterine tumors. Hexokinase ii is an enzyme central to glucose metabolism and glucose repression in the yeast saccharomyces cerevisiae. Hexokinase i sets the pace of glycolysis in the brain, catalyzing the atpdependent phosphorylation of glucose.
Molecular complex of three testisspecific isozymes associated with the mouse sperm fibrous sheath. Walkeron the presence of two soluble glucosephosphorylating enzymes in adult. The enzyme activities and isozyme distribution of the three glycolytic regulator enzymes hexokinase, phosphofructokinase and pyruvate kinase were studied in. The study reveals that hkii detects glucose depletion to adapt to metabolic suppression by facilitating autophagy. Get a printable copy pdf file of the complete article 462k, or click on a. Binding of noncatalytic atp to human hexokinase i highlights the. Cancers doubleedged sword acting as both facilitator and gatekeeper of malignancy when bound to mitochondria skip to main content thank you for visiting. The elevated rates of glucose metabolism, tumor progression, and the high patient mortality has been predominantly associated with the overexpression of hk2 in different cancers. Hexokinase ii is the predominant isoform in insulinsensitive tissues and. Multiple forms of enzymes appearing as artifacts of purification studies with ribonuclease 42 andglyceraldehyde phosphate dehydrogenase 4 have shown that multiple forms of an enzyme can appear. The hexokinase hk of the human red blood cell rbc was separated into two distinct major isozymes by fast protein liquid chromatography using a linear salt gradient on a monoq column.
The molecular bases for the testisspecific expression are diverse. Moreover, because of the presence of glucose in the crystallization buffer and in the. Glucokinase gk is a hexokinase isozyme, related homologously to at least three other hexokinases. The yeast hexokinase represents a good example of multiple forms in which the only differences that can be detected are charge differences. Difference in kinetic properties between hexokinase type i isoenzymes from various. In the presence of a competitive inhibitor, an enzyme will have the same. Reported here is the structure of a new crystal form of recombinant human hexokinase i, which complements existing crystal structures. There was an inverse correlation between the expression of hk1 and hk2 in.
225 1153 642 590 1384 415 1362 242 619 527 991 251 453 170 1430 922 230 114 1499 1053 1362 27 742 67 464 464 836 1204 474 1497 686 110