Hexokinase Family Research Articles

Evolutionary Lineages and Functional Diversification of Plant Hexokinases

Sequencing data from 10 species show that a plant hexokinase (HXK) family contains 5-11 genes. Functionally, a given family can include metabolic catalysts, glucose signaling proteins, and non-catalytic, apparent regulatory enzyme homologs. This study has two goals. The first aim is to develop a predictive method to determine which HXK proteins within a species have which type of function. The second aim is to determine whether HXK-dependent glucose signaling proteins occur among more primitive plants, as well as among angiosperms. Using a molecular phylogeny approach, combined with selective experimental testing, we found that non-catalytic HXK homologs might occur in all plants, including the relatively primitive Selaginella moellendorffi. We also found that different lineages of angiosperm HXKs have apparent conserved features for catalytic activity and for sub-cellular targeting. Most higher-plant HXKs are expressed predominantly at mitochondria, with HXKs of one lineage occurring in the plastid, and HXKs of one monocot lineage occurring in the cytosol. Using protoplast transient expression assays, we found that HXK glucose signaling proteins occur likely in all higher plants and in S. moellendorffi as well. Thus, the use of glucose by plant HXK isoforms in metabolism and/or as a regulatory metabolite occurs as widespread, conserved processes.

Medicinal Chemistry Approaches for Glucokinase Activation to Treat Type 2 Diabetes

Type 2 Diabetes (T2D), characterized by elevated levels of blood glucose is a complex disease mainly caused by defects in hepatic glucose balance and the failure of pancreatic beta-cells to secrete enough insulin to overcome insulin resistance. Glucokinase (GK) is a member of hexokinase family of enzymes that are responsible for the phosphorylation of glucose to glucose-6-phosphate for further utilization in cells. It plays a key role in glucose homeostasis in cells that express this enzyme, such as beta-cells and hepatocytes. It promotes glycogen synthesis in the liver and glucose-sensitive insulin release in the beta-cell. While hypoglycemia due to the increased insulin secretion could be a potential concern it was hypothesized that a GK activator with optimized properties would be able to both blunt the postprandial glucose excursion and lower the fasting blood glucose in T2D patients. As a result of intensive medicinal chemistry efforts a number of small molecules have been discovered as GK activators many of which showed antidiabetic effects in animal models of T2D. Some of these activators have advanced into human clinical studies. With the promising preclinical data in hand, GK activators represent a promising and new treatment option for T2D.

Recent advances in glucokinase activators for the treatment of type 2 diabetes

Glucokinase is a member of the hexokinase family of enzymes that are responsible for the phosphorylation of glucose to glucose-6-phosphate for further utilization in cells. The enzyme plays a key role in glucose homeostasis. Phosphorylation of glucose by glucokinase in the liver promotes glycogen synthesis, while in the beta-cell it results in insulin release. Activators of glucokinase increase the sensitivity of the enzyme to glucose, leading to increased insulin secretion and liver glycogen synthesis and a decrease in liver glucose output. Thus, small molecule glucokinase activators have been demonstrated to be effective glucose-lowering agents in animal models of type 2 diabetes and have advanced into clinical studies.

Crystal structure of yeast hexokinase PI in complex with glucose: A classical “induced fit” example revised

Hexokinase is the first enzyme in the glycolytic pathway that catalyzes the transfer of a phosphoryl group from ATP to glucose to form glucose-6-phosphate and ADP. Two yeast hexokinase isozymes are known, namely PI and PII. Here we redetermined the crystal structure of yeast hexokinase PI from Saccharomyces cerevisiae as a complex with its substrate, glucose, and refined it at 2.95 A resolution. Comparison of the holo-PI yeast hexokinase and apo-hexokinase structures shows in detail the rigid body domain closure and specific loop movements as glucose binds and sheds more light on structural basis of the "induced fit" mechanism of reaction in the HK enzymatic action. We also performed statistical coupling analysis of the hexokinase family, which reveals two co-evolved continuous clusters of amino acid residues and shows that the evolutionary coupled amino acid residues are mostly confined to the active site and the hinge region, further supporting the importance of these parts of the protein for the enzymatic catalysis.

Glucose Phosphorylation and Mitochondrial Binding Are Required for the Protective Effects of Hexokinases I and II

Alterations in glucose metabolism have been demonstrated for diverse disorders ranging from heart disease to cancer. The first step in glucose metabolism is carried out by the hexokinase (HK) family of enzymes. HKI and II can bind to mitochondria through their N-terminal hydrophobic regions, and their overexpression in tissue culture protects against cell death. In order to determine the relative contributions of mitochondrial binding and glucose-phosphorylating activities of HKs to their overall protective effects, we expressed full-length HKI and HKII, their truncated proteins lacking the mitochondrial binding domains, and catalytically inactive proteins in tissue culture. The overexpression of full-length proteins resulted in protection against cell death, decreased levels of reactive oxygen species, and possibly inhibited mitochondrial permeability transition in response to H(2)O(2). However, the truncated and mutant proteins exerted only partial effects. Similar results were obtained with primary neonatal rat cardiomyocytes. The HK proteins also resulted in an increase in the phosphorylation of voltage-dependent anion channel (VDAC) through a protein kinase Cepsilon (PKCepsilon)-dependent pathway. These results suggest that both glucose phosphorylation and mitochondrial binding contribute to the protective effects of HKI and HKII, possibly through VDAC phosphorylation by PKCepsilon.

Oncogenic H-Ras increases glucose metabolism with an induction of glucose transporter GLUT1 and hexokinase 2 expression

INTRODUCTION: Oncogenic Ras supports the development of cancer cells by activating pathways that stimulate growth and prevent apoptosis. Increased glucose metabolism is a hallmark of cellular transformation. The profiles of glucose transporter (GLUT) and/or hexokinase (HK) family members may be altered to more efficiently utilize glucose after mutant Ras transformation. HK1 and 2 mitochondrial localization have been associated with alterations in apoptosis sensitivities.

Molecular and biochemical characterization of novel glucokinases from Trypanosoma cruzi and Leishmania spp.

Glucokinase genes, found in the genome databases of Trypanosoma cruzi and Leishmania major, were cloned and sequenced. Their expression in Escherichia coli resulted in the synthesis of soluble and active enzymes, TcGlcK and LmjGlcK, with a molecular mass of 43kDa and 46kDa, respectively. The enzymes were purified, and values of their kinetic parameters determined. The Km values for glucose were 1.0mM for TcGlcK and 3.3mM for LmjGlcK. For ATP, the Km values were 0.36mM (TcGlcK) and 0.35mM (LmjGlcK). A lower Km value for glucose (2.55mM) was found when the (His)6-tag was removed from the recombinant LmjGlcK, whereas the TcGlcK retained the same value. The Vmax's of the T. cruzi and L. major GlcKs were 36.3 and 30.9U/mg of protein, respectively. No inhibition was exerted by glucose-6-phosphate. Similarly, no inhibition by inorganic pyrophosphate was found in contrast to previous observations made for the T. cruzi and L. mexicana hexokinases. Both trypanosomatid enzymes were only able to phosphorylate glucose indicating that they are true glucokinases. Gel-filtration chromatography showed that the GlcK of both trypanosomatids may occur as a monomer or dimer, dependent on the protein concentration. Both GlcK sequences have a type-1 peroxisome-targeting signal. Indeed, they were shown to be present inside glycosomes using three different methods. These glucokinases present highest, albeit still a moderate 24% sequence identity with their counterpart from Trichomonas vaginalis, which has been classified into group A of the hexokinase family. This group comprises mainly eubacterial and cyanobacterial glucokinases. Indeed, multiple sequence comparisons, as well as kinetic properties, strongly support the notion that these trypanosomatid enzymes belong to group A of the hexokinases, in which they, according to a phylogenetic analysis, form a separate cluster.

Crystal Structures of an ATP-dependent Hexokinase with Broad Substrate Specificity from the Hyperthermophilic Archaeon Sulfolobus tokodaii

Hexokinase catalyzes the phosphorylation of glucose to glucose 6-phosphate by using ATP as a phosphoryl donor. Recently, we identified and characterized an ATP-dependent hexokinase (StHK) from the hyperthermophilic archaeon Sulfolobus tokodaii, which can phosphorylate a broad range of sugar substrates, including glucose, mannose, glucosamine, and N-acetylglucosamine. Here we present the crystal structures of StHK in four different forms: (i) apo-form, (ii) binary complex with glucose, (iii) binary complex with ADP, and (iv) quaternary complex with xylose, Mg(2+), and ADP. Forms i and iii are in the open state, and forms ii and iv are in the closed state, indicating that sugar binding induces a large conformational change, whereas ADP binding does not. The four different crystal structures of the same enzyme provide "snapshots" of the conformational changes during the catalytic cycle. StHK exhibits a core fold characteristic of the hexokinase family, but the structures of several loop regions responsible for substrate binding are significantly different from those of other known hexokinase family members. Structural comparison of StHK with human N-acetylglucosamine kinase and other hexokinases provides an explanation for the ability of StHK to phosphorylate both glucose and N-acetylglucosamine. A Mg(2+) ion and coordinating water molecules are well defined in the electron density of the quaternary complex structure. This structure represents the first direct visualization of the binding mode for magnesium to hexokinase and thus allows for a better understanding of the catalytic mechanism proposed for the entire hexokinase family.

Effects of Restricted Feeding on Daily Fluctuations of Hepatic Functions Including P450 Monooxygenase Activities in Rats

Hepatic P450 monooxygenase activities, assessed by measurement of 7-alkoxycoumarin O-dealkylase (ACD) activities, show obvious daily fluctuations in male rats with high values during the dark period and low values during the light period. We have already confirmed that the ACD activities are controlled by the suprachiasmatic nucleus (SCN), which is well known as the oscillator of circadian rhythm. Recently, it is reported that circadian oscillators exist not only in the SCN but also in peripheral organs. To date, it is unclear which circadian oscillators predominantly drive the daily fluctuations of hepatic ACD activities. To address this question, we examined the effects of restricted feeding, which uncouples the circadian oscillators in the liver from the central pacemaker in the SCN, on the daily fluctuations in hepatic ACD activities in male rats. Here we show that restricted feeding inverts the oscillation phase of the daily fluctuations in hepatic ACD activities. Regarding the hepatic P450 content, there were no fluctuations between the light and dark periods under ad libitum and restricted feeding conditions. Therefore, it is considered that the daily fluctuations in hepatic ACD activities are predominantly driven by the circadian factors in peripheral organs rather than by the oscillator in the SCN directly.

Hypothesis: Structures, evolution, and ancestor of glucose kinases in the hexokinase family

Glucose kinase, which we tentatively use in this review, represents the enzymes catalyzing the phosphorylation of glucose and other hexoses by means of phosphoryl donors (ATP, ADP, and inorganic polyphosphate [poly(P)]). Except for glucose kinases utilizing ADP, all other glucose kinases belong to the hexokinase (HK) family and are classified into three groups based on primary structural information, i.e., groups HK, A, and B. The structural and evolutionary relationships of glucose kinases belonging to the above three groups have been controversial due to the lack of tertiary structural information on those in groups A and B. However, recent studies on the tertiary structures of poly(P)/ATP-glucomannokinase (GMK: a glucose kinase in group B) from Arthrobacter sp. strain KM and glucokinase (GK) (ecoGK: a glucose kinase in group A) from Escherichia coli have shed light on this problem. A comparison of the tertiary structures of GMK and ecoGK with those of glucose kinases in group HK demonstrated that both GMK and ecoGK are structurally homologous with glucose kinases in group HK, and that glucose kinases belonging to groups HK, A, and B in the HK family evolved divergently from a common ancestor. Based on the simple structure of GMK compared to those of ecoGK and glucose kinases in group HK, and the putative poly(P)-binding site in GMK, we propose that the ancestor of glucose kinases in the HK family was similar to GMK and used poly(P). We also discuss the ancestor and evolutionary process of ROK proteins, whose primary structures are homologous with those of glucose kinases in group B, in connection with the ancestor and evolutionary process of glucose kinases in the HK family.

Oxygen-Dependent Regulation of Hepatic Glucose Metabolism

This chapter discusses the oxygen-dependent regulation of hepatic glucose metabolism. Glucose is converted to glucose 6-phosphate by hexokinases, which exist in several isoforms. The glucokinase (GK) represents the major active form of the hexokinases in the liver, whereas hexokinases I–III are operative in the other majororgans. GK expression is restricted to hepatocytes, pancreatic β cells, and some neuroendocrine cells of the gastrointestinal tract. Contrary to other hexokinase family members, the GK has a lower affinity for glucose with sigmoidal kinetics and is not inhibited by its reaction product glucose 6-phosphate. In this study, protocols allowing the measurement of metabolic activities under the control of O 2 , as well as studying the modulatory role of O 2 for gene expression in primary hepatocytes are discussed. Degradation of glucose to acetyl-CoA, the synthesis and degradation of glycogen stores is analyzed. Metabolic zonation of liver, oxygen gradients, and modulation of carbohydrate metabolism by O 2 is elaborated. Preparation and culture of primary hepatocytes is discussed. Transfection of hepatocytes using the calcium-phosphate precipitation and luciferase assay is also described.

The Hexokinase of the Hyperthermophile Thermoproteus tenax: ATP-DEPENDENT HEXOKINASES AND ADP-DEPENDENT GLUCOKINASES, TWO ALTERNATIVES FOR GLUCOSE PHOSPHORYLATION IN ARCHAEA

The phosphorylation of glucose by different sugar kinases plays an essential role in Archaea because of the absence of a phosphoenolpyruvate-dependent transferase system characteristic for Bacteria. In the genome of the hyperthermophilic Archaeon Thermoproteus tenax a gene was identified with sequence similarity to glucokinases of the so-called ROK family (repressor protein, open reading frame, sugar kinase). The T. tenax enzyme, like the recently described ATP-dependent "glucokinase" from Aeropyrum pernix, shows the typical broad substrate specificity of hexokinases catalyzing not only phosphorylation of glucose but also of other hexoses such as fructose, mannose, or 2-deoxyglucose, and thus both enzymes represent true hexokinases. The T. tenax hexokinase shows strikingly low if at all any regulatory properties and thus fulfills no important control function at the beginning of the variant of the Embden-Meyerhof-Parnas pathway in T. tenax. Transcript analyses reveal that the hxk gene of T. tenax is cotranscribed with an upstream located orfX, which codes for an 11-kDa protein of unknown function. Growth-dependent studies and promoter analyses suggest that post-transcriptional RNA processing might be involved in the generation of the monocistronic hxk message, which is observed only under heterotrophic growth conditions. Data base searches revealed T. tenax hexokinase homologs in some archaeal, few eukaryal, and many bacterial genomes. Phylogenetic analyses confirm that the archaeal hexokinase is a member of the so-called ROK family, which, however, should be referred to as ROK group because it represents a group within the bacterial glucokinase fructokinase subfamily II of the hexokinase family. Thus, archaeal hexokinases represent a second major group of glucose-phosphorylating enzymes in Archaea beside the recently described archaeal ADP-dependent glucokinases, which were recognized as members of the ribokinase family. The distribution of the two types of sugar kinases, differing in their cosubstrate as well as substrate specificity, within Archaea is discussed on the basis of physiological constraints of the respective organisms.

The tomato hexokinase LeHXK1 cloning, mapping, expression pattern and phylogenetic relationships

A tomato ( Lycopersicon esculentum L. Mill.) hexokinase, LeHXK1, was cloned from a tomato fruit cDNA library. Functional complementation of triple-mutant yeast cells ( hxk1 hxk2 glk1) that lack the ability to phosphorylate glucose and fructose confirmed the identity of LeHXK1 as a hexokinase. LeHXK1 was mapped to tomato chromosome 3-distinct from LeHXK2 which mapped to chromosome 6. Interestingly, expression of LeHXK1 was very low in leaves and very high in young tomato fruits. Two hexokinase isozymes, HXKl and HXK2, were chromatographically separated from young fruit extracts, Antisense StHXK1 the homologue of, LeHXK1, knocked out the HXKl isozyme, identifying HXKl as the gene product of LeHXK1. Phylogenetic analysis of eukaryotic and prokaryotic hexose phosphorylating enzymes indicates that tomato hexokinase genes, LeHXK1 and LeHXK2, and the known eukaryotic hexokinases and ‘glucokinases’ belong to the same hexokinase family whose members are unrelated to the glucose-specific glucokinases of bacteria. Hence, the data suggest that thus far no true eukaryotic glucokinases have been identified, neither from plants, nor from fungi or mammalian cells.

Unique phylogenetic relationships of glucokinase and glucosephosphate isomerase of the amitochondriate eukaryotes Giardia intestinalis, Spironucleus barkhanus and Trichomonas vaginalis

Glucokinase (GK) and glucosephosphate isomerase (GPI), the first two enzymes of the glycolytic pathway of the diplomonads Giardia intestinalis and Spironucleus barkhanus, Type I amitochondriate eukaryotes, were sequenced. GPI of the parabasalid Trichomonas vaginalis was also sequenced. The diplomonad GKs belong to a family of specific GKs present in cyanobacteria, in some proteobacteria and also in T. vaginalis, a Type II amitochondriate protist. These enzymes are not part of the hexokinase family, which is broadly distributed among eukaryotes, including the Type I amitochondriate parasite Entamoeba histolytica. G. intestinalis GK expressed in Escherichia coli was specific for glucose and glucosamine, as are its eubacterial homologs. The sequence of diplomonad and trichomonad GPIs formed a monophyletic group more closely related to cyanobacterial and chloroplast sequences than to cytosolic GPIs of other eukaryotes and prokaryotes. The findings show that certain enzymes of the energy metabolism of these amitochondriate protists originated from sources different than those of other eukaryotes. The observation that the two diplomonads and T. vaginalis share the same unusual GK and GPI is consistent with gene trees that suggest a close relationship between diplomonads and parabasalids. The intriguing relationships of these enzymes to cyanobacterial (and chloroplast) enzymes might reflect horizontal gene transfer between the common ancestor of the diplomonad and parabasalid lineages and the ancestor of cyanobacteria.

Biochemical characterization, cloning, and sequencing of ADP-dependent (AMP-forming) glucokinase from two hyperthermophilic archaea, Pyrococcus furiosus and Thermococcus litoralis.

The ADP-dependent (AMP-forming) glucokinases from the hyperthermophilic archaea Pyrococcus furiosus and Thermococcus litoralis catalyze the phosphorylation of glucose using ADP as the essential phosphoryl group donor. Both enzymes were purified to homogeneity and characterized with regard to each other. The enzymes had similar enzymological properties as to substrate specificity, coenzyme specificity, optimum pH, and thermostability. However, a difference was observed in the subunit composition; while the T. litoralis enzyme is a monomer with a molecular mass of 52 kDa, the P. furiosus enzyme has a molecular mass of about 100 kDa and consists of two subunits with identical molecular masses of 47 kDa. The genes encoding these enzymes were cloned and sequenced. The gene for the P. furiosus enzyme contains an open reading frame for 455 amino acids with a molecular weight of 51,265, and that for the T. litoralis enzyme contains an open reading frame for 467 amino acids with a molecular weight of 53,621. About 59% similarity in amino acid sequence was observed between these two enzymes, whereas they did not show similarity with any ATP-dependent kinases that have been reported so far. In addition, two phosphate binding domains, and adenosine and glucose binding motifs commonly conserved in the eukaryotic hexokinase family were not observed.

Differential Metabolic Effects of Adenovirus-mediated Glucokinase and Hexokinase I Overexpression in Rat Primary Hepatocytes

The first step of glucose metabolism is the phosphorylation of glucose, catalyzed by the hexokinase family of enzymes. To address the metabolic impact of increasing glucose phosphorylation capacity in liver, rat primary hepatocytes were treated with recombinant adenoviruses containing the cDNAs encoding either rat liver glucokinase (AdCMV-GKL) or rat hexokinase I (AdCMV-HKI). Maximal glucose phosphorylation in AdCMV-GKL- and AdCMV-HKI-treated hepatocytes was increased 7.1 +/- 1.2- and 6.3 +/- 0.8-fold, respectively, over hepatocytes treated with an adenovirus expressing beta-galactosidase. Glucose usage (measured with 3 and 20 m 2-[3H]glucose and 5-[3H]glucose) was significantly increased in AdCMV-GKL-treated cells preincubated in 1 or 25 mM glucose. Treatment of hepatocytes with AdCMV-HKI also caused enhanced glucose utilization, but the increases were smaller and were less apparent in cells preincubated in high (25 mM) glucose. AdCMV-GKL-treated hepatocytes incubated for 48 h in the presence of variable glucose concentrations had glycogen levels that were maximally 15.0 +/- 0. 6-fold greater than levels in corresponding control cells. AdCMV-HKI-treated hepatocytes incubated under similar conditions had unchanged glycogen levels relative to controls. In AdCMV-GKL-treated cells, lactate output was increased to a maximum of 3.0 +/- 0.4-fold (at 25 mM glucose), glucose oxidation was increased 3.5 +/- 0.3-fold, and triglyceride production was unchanged relative to untreated cells. Among these three parameters, only lactate production was increased in AdCMV-HKI-treated cells, and then only at low glucose concentrations. We conclude that overexpression of glucokinase has potent effects on glucose storage and utilization in hepatocytes and that these effects are not matched by overexpression of hexokinase I.

Cloning, Expression, and Characterization of Polyphosphate Glucokinase from Mycobacterium tuberculosis

Polyphosphate glucokinase from Mycobacterium tuberculosis catalyzes the phosphorylation of glucose using polyphosphate or ATP as the phosphoryl donor. The M. tuberculosis H37Rv gene encoding this enzyme has been cloned, sequenced, and expressed in Escherichia coli. The gene contains an open reading frame for 265 amino acids with a calculated mass of 27,400 daltons. The recombinant polyphosphate glucokinase was purified 189-fold to homogeneity and shown to contain dual enzymatic activities, similar to the native enzyme from H37Ra strain. The high G+C content in the codon usage (64.5%) of the gene and the absence of an E. coli-like promoter consensus sequence are consistent with other mycobacterial genes. Two phosphate binding domains conserved in the eukaryotic hexokinase family were identified in the polyphosphate glucokinase sequence, however, "adenosine" and "glucose" binding motifs were not apparent. In addition, a putative polyphosphate binding region is also proposed for the polyphosphate glucokinase enzyme.

Functional Organization of Mammalian Hexokinase II: RETENTION OF CATALYTIC AND REGULATORY FUNCTIONS IN BOTH THE NH2- AND COOH-TERMINAL HALVES

The mammalian hexokinase (HK) family includes three closely related 100-kDa isoforms (HKI-III) that are thought to have arisen from a common 50-kDa precursor by gene duplication and tandem ligation. Previous studies of HKI indicated that a glucose 6-phosphate (Glu-6-P)-regulated catalytic site resides in the COOH-terminal half of the molecule and that the NH2-terminal half contains only a Glu-6-P binding site. In contrast, we now show that proteins representing both halves of human and rat HKII have catalytic activity and that each is inhibited by Glu-6-P. The intact enzyme and the NH2- and COOH-terminal halves of the enzyme each increase glucose utilization when expressed in Xenopus oocytes. Mutations corresponding to either Asp-209 or Asp-657 in the intact enzyme completely inactivate the NH2- and COOH-terminal half enzymes, respectively. Mutation of either of these sites results in a 50% reduction of activity in the 100-kDa enzyme. Mutation of both sites results in a complete loss of activity. This suggests that each half of the HKII molecule retains catalytic activity within the 100-kDa protein. These observations indicate that HKI and HKII are functionally distinct and have evolved differently.

Convergent evolution of similar enzymatic function on different protein folds: the hexokinase, ribokinase, and galactokinase families of sugar kinases.

Kinases that catalyze phosphorylation of sugars, called here sugar kinases, can be divided into at least three distinct nonhomologous families. The first is the hexokinase family, which contains many prokaryotic and eukaryotic sugar kinases with diverse specificities, including a new member, rhamnokinase from Salmonella typhimurium. The three-dimensional structure of hexokinase is known and can be used to build models of functionally important regions of other kinases in this family. The second is the ribokinase family, of unknown three-dimensional structure, and comprises pro- and eukaryotic ribokinases, bacterial fructokinases, the minor 6-phosphofructokinase 2 from Escherichia coli, 6-phosphotagatokinase, 1-phosphofructokinase, and, possibly, inosine-guanosine kinase. The third family, also of unknown three-dimensional structure, contains several bacterial and yeast galactokinases and eukaryotic mevalonate and phosphomevalonate kinases and may have a substrate binding region in common with homoserine kinases. Each of the three families of sugar kinases appears to have a distinct three-dimensional fold, since conserved sequence patterns are strikingly different for the three families. Yet each catalyzes chemically equivalent reactions on similar or identical substrates. The enzymatic function of sugar phosphorylation appears to have evolved independently on the three distinct structural frameworks, by convergent evolution. In addition, evolutionary trees reveal that (1) fructokinase specificity has evolved independently in both the hexokinase and ribokinase families and (2) glucose specificity has evolved independently in different branches of the hexokinase family. These are examples of independent Darwinian adaptation of a structure to the same substrate at different evolutionary times. The flexible combination of active sites and three-dimensional folds observed in nature can be exploited by protein engineers in designing and optimizing enzymatic function.

Expression and site-directed mutagenesis of hepatic glucokinase

Soluble rat liver glucokinase was expressed at high levels at 22 degrees C in the BL21(DE3)pLysS strain of Escherichia coli. Aspartate-211 of yeast hexokinase has been implicated as a catalytic residue from crystallographic data. The corresponding residue in rat liver glucokinase, aspartate-205, was mutated to alanine and the expressed mutant had 1/500th of the activity of the wild type, with no change in the Km values for glucose or ATP. The results support a role for this residue as a base catalyst in the glucokinase reaction and, most probably, a similar role in the reactions of all members of the hexokinase family.