Kinetic and regulatory properties of HK I(+), a modified form of the type I isozyme of mammalian hexokinase in which interactions between the N- and C-terminal halves have been disrupted

A modified form (HK I(+)) of rat Type I hexokinase (HK I) has been expressed. HK I(+) contains a centrally located polyalanine insert which, along with the known helical propensity of adjacent sequence, was expected to lead to alpha-helix formation, with resulting distension of the molecule and disruption of interactions between the N- and C-terminal halves. The properties of HK I(+) are consistent with this expectation and with previous proposals that (1) inhibition of HK I by Glc-6-P or its analogs and antagonism of this inhibition by P(i) result from competition of these ligands for a binding site in the N-terminal half of HK I, with resulting conformational changes propagated through interactions with the catalytic C-terminal half, and (2) binding of Glc-6-P to a site in the C-terminal half of HK I is obstructed by interactions between the halves, present in HK I but not HK I(+).     

Complete amino acid sequence of the type III isozyme of rat hexokinase, deduced from the cloned cDNA

Clones containing cDNA coding for the Type III isozyme of rat hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) were isolated from a library prepared in lambda gt10 with rat liver mRNA. Three clones were characterized. Their composite sequence includes the entire coding region for Type III hexokinase, 3' untranslated sequence extending into the polyadenylated region, and 80 bp of 5' untranslated sequence. Extensive similarity in sequence of N- and C-terminal halves of the enzyme, previously seen with the Type I isozyme, is consistent with the view that these 100-kDa mammalian hexokinases are the evolutionary result of duplication and fusion of a gene coding for an ancestral hexokinase having a molecular weight of approximately 50 kDa. Extensive similarities are seen between sequences of the Type I and III isozymes, and those reported for mammalian glucokinase (also called Type IV hexokinase) and for the hexokinase and glucokinase of yeast. Residues thought to be involved in catalytic function are highly conserved in all of these enzymes. Based on a quantitative comparison of sequence similarities, it is concluded that the 50-kDa mammalian glucokinase is more closely related to the 100-kDa mammalian enzymes than it is to the 50-kDa enzymes from yeast. One interpretation of this might be that the mammalian glucokinase arose by resplitting of the gene coding for the 100-kDa mammalian hexokinases.     

Functional organization and evolution of mammalian hexokinases: mutations that caused the loss of catalytic activity in N-terminal halves of type I and type III isozymes.

Mammalian hexokinases are believed to have evolved from a 100-kDa hexokinase which itself is a product of duplication and fusion of an ancestral gene encoding a 50-kDa glucose 6-phosphate-sensitive hexokinase. Type II hexokinase has been shown to possess two distinct functional active sites, one in each half, which functionally resemble the original 100-kDa hexokinase, whereas type I and III isozymes possess only one active site in the C-terminal halves. This study was conducted to identify which mutations caused the loss of catalytic activity in the N-terminal halves of type I and III isozymes. Arg 174 and Ser 447 in type I isozyme and Asp 244 in type III isozyme are speculated to be the cause, because they reside adjacent to the "catalytic" site and corresponding residues, Gly 174, Asp 447, and Gly 231, are conserved in the N-terminal half of type II isozyme as well as all other 50-kDa units that possess catalytic activity. Mutations G174R and D447S in the N-terminal half of type II isozyme reduced specific activity by approximately 79 and 57%, respectively. Therefore, neither mutation alone can account for the inactivation of the N-terminal active site in type I isozyme. Either mutation, G174R or D447S, had moderate effects on Michaelis constants, K(m), for glucose and ATP. Mg(2+). Intriguingly, mutation D447S introduced a novel inhibition by unchelated ATP (K(i) = 68 microM ATP, competitive vs ATP. Mg(2+)) to the N-terminal active site of type II isozyme. Mutation G231D caused instability to type II hexokinase and near complete loss of catalytic activity (95%), suggesting that mutation G231D not only hinders catalysis at the N-terminal active site but also leads to structural instability in type II hexokinase.     

Membrane potential-dependent conformational changes in mitochondrially bound hexokinase of brain

Previously characterized monoclonal antibodies (Mabs) were used in a study of Type I hexokinase from rat brain. Based on the relative reactivity of these Mabs with soluble and mitochondrially bound forms, binding to mitochondria was shown to affect specific epitopic regions in both N- and C-terminal halves of the enzyme and to modulate conformational changes induced by binding of the ligands, Glc or ATP. Reactivities with Mabs recognizing epitopes in two defined regions of the N-terminal half and one defined region of the C-terminal half of the mitochondrially bound enzyme were selectively affected by mitochondrial membrane potential, or by addition of oligomycin, carboxyatractyloside, or bongkrekic acid. The Glc-6-P analog, 1 ,5-anhydroglucitol-6-P, was much more effective as a competitive inhibitor against extramitochondrial ATP than against intramitochondrial ATP generated by oxidative phosphorylation. These results provide further insight into the role of hexokinase-mitochondrial interactions in regulation of cerebral glucose metabolism.     

Chimeric phosphofructokinases involving exchange of the N- and C-terminal halves of mammalian isozymes: implications for ligand binding sites

Two phosphofructokinase (PFK) chimeras were constructed by exchanging the N- and C-terminal halves of the mammalian M- and C-type isozymes, to investigate the contribution of each terminus to the catalytic site and the fructose-2,6-P(2)/fructose-1,6-P(2) allosteric site. The homogeneously-purified chimeric enzymes organized into tetramers, and exhibited kinetic properties for fructose-6-P and MgATP similar to those of the native enzyme that furnished the N-terminal domain in each case, whereas their fructose-2,6-P(2) activatory characteristics coincided with those of the isozyme that provided the C-terminal half. This reflected the role of each domain in the formation of the corresponding binding site. Grafting the N-terminus of PFK-M onto the C-terminus of the fructose-1,6-P(2) insensitive PFK-C restored transduction of this signal to the catalytic site, which significance is also discussed.     

Function of interdomain α-helix in human brain hexokinase: covalent linkage and catalytic regulation between N- and C-terminal halves

Human brain relies on a steady supply of glucose as the source of fuel, and type I hexokinase is the major isozyme governing the introduction of glucose to glycolysis in the brain. One unique regulatory property associated with type I isozyme is the alleviation of product inhibition by inorganic phosphate which binds to the N-terminal half, and the conformational change induced by inorganic phosphate must be propagated to the active site in the C-terminal half. With a single interdomain α-helix as the only covalent connection between the N- and C-terminal halves, the question arises as what role the interdomain α-helix plays at the interdomain signal transduction. Two mutants were constructed in an attempt to answer this question. The first mutant, A464P/E465G, with a helix breaker embedded in the interdomain α-helix had a smaller magnitude of phosphate alleviation than the wild type. The second mutant, with an insertion of seven additional residues between Gln 466 and His 467, had this phosphate relief property further diminished. Neither mutant showed dramatic changes nor the other kinetic properties. It is speculated that the interdomain α-helix is important for keeping the proper non-covalent contact so that transmission of the conformational changes across the N- and C-terminal half boundary can be achieved.     

Bovine hexokinase type I: Full-length cDNA sequence and characterisation of the recombinant enzyme

This study reports the revised and full-length cDNA sequence of bovine hexokinase type I obtained from bovine brain. Since dissimilarities have been observed between the published bovine hexokinase type I coding sequence (GenBank accession no. M65140) (Genomics 11: 1014-1024, 1991) and an analysed portion of bovine hexokinase type I gene, the entire open reading frame was re-sequenced and the ends of cDNA isolated by rapid amplification of cDNA ends. The coding sequences, when compared with the published bovine hexokinase type I, contained a large number of mismatches that lead to changes in the resulting amino acid sequence. The revisions result in a hexokinase type I cDNA of 3619 bp that encodes a protein of 917 amino acids highly homologous to human hexokinase type I. The expression of the recombinant full-length enzyme demonstrated that it was a catalytically active hexokinase. When characterised for its kinetic and regulatory properties, it displayed the same affinity for glucose and MgATP as the human hexokinase type I and was inhibited by glucose 6-phosphate competitively versus MgATP. The production of the N- and C-terminal recombinant halves of the enzyme followed by comparison with the full-length hexokinase indicated that the catalytic activity is located in the C-terminal domain. (Mol Cell Biochem 268: 9–18, 2005)     

Complete amino acid sequence of the type II isozyme of rat hexokinase, deduced from the cloned cDNA: comparison with a hexokinase from novikoff ascites tumor

The 917-residue amino acid sequence of the Type II isozyme of rat hexokinase has been deduced from the nucleotide sequence of cloned cDNA. The sequences of 197 nucleotides in the 5' untranslated region and 687 bases of the 3' untranslated region have also been determined. A region of overlap between two discrete cDNA clones was confirmed by isolation and sequencing of a genomic DNA clone that spanned the region. Within this region, the 634-nucleotide coding sequence was divided into three exons, each of 150-250 nucleotides; these results suggest that the gene encoding Type II hexokinase is likely to be quite complex. There is extensive similarity between the sequences of the N- and C-terminal halves of the Type II isozyme, as previously seen with the Type I and III isozymes; this is consistent with the view that these enzymes evolved by a process of gene duplication and fusion. A cDNA encoding the entire C-terminal half of a hexokinase from Novikoff ascites tumor cells was also isolated and found to be identical to a cDNA encoding the corresponding region of the Type II isozyme of skeletal muscle. Northern analysis indicated that a single mRNA, approx 5200 nucleotides in length, encoded both the skeletal muscle and the tumor enzymes. These results do not support previous speculation that the hexokinase isozymes of normal tissue are distinct from those of tumors, and suggest the possibility that post-translational modifications of a single protein species might account for apparent differences between the isozymes of normal and tumor tissues.     

Rat brain hexokinase: location of the allosteric regulatory site in a structural domain at the N-terminus of the enzyme

After denaturation in 0.6 M guanidine hydrochloride, rat brain hexokinase becomes highly susceptible to proteolysis by trypsin. Glucose 6-phosphate (Glc-6-P) and its analog, 1,5-anhydroglucitol 6-phosphate, selectively protect the N-terminal half of the molecule from proteolysis. These compounds do not protect the C-terminal half of the molecule, nor do they protect enzyme activity; the Glc analog, N-acetylglucosamine, does protect the C-terminal domain and catalytic activity, but does not prevent proteolysis of the N-terminal half of the molecule. These results are consistent with previous work [M. Nemat-Gorgani and J. E. Wilson (1986) Arch. Biochem. Biophys. 251, 97-103; D. M. Schirch and J. E. Wilson (1987) Arch. Biochem. Biophys. 254, 385-396] demonstrating that binding sites for both hexose and nucleotide substrates, and thus catalytic function, are associated with a 40-kDa domain located at the C-terminus of the enzyme. They further demonstrate that the binding site for the allosteric effector, Glc-6-P, lies in the N-terminal half of the molecule and is distinct from the catalytic site. Using protection against proteolysis as a reflection of binding, it is shown that the Glc-6-P binding site in the N-terminal region has all the characteristics described for the allosteric effector site on this enzyme in terms of affinity for Glc-6-P, specificity, and synergistic interactions with the hexose binding site in the C-terminal region of the molecule. This disposition of catalytic and regulatory functions in discrete halves of the molecule is consistent with suggestions by several investigators that mammalian hexokinases evolved by a process of duplication and fusion of an ancestral gene coding for a hexokinase similar to the present-day yeast enzyme, with the regulatory site of mammalian hexokinases having evolved from what was originally a catalytic site.     

Isolation and characterization of the discrete N- and C-terminal halves of rat brain hexokinase: retention of full catalytic activity in the isolated C-terminal half

Selective stabilization of either the N- or C-terminal half (by ligands binding to these regions) of rat brain hexokinase against partial denaturation with guanidine hydrochloride and subsequent digestion with trypsin has provided a means for isolating these regions, referred to as N fragment and C fragment, respectively, in quantities adequate for characterization. The N fragment (mol wt 52 kDa) is devoid of catalytic activity. In contrast, the C fragment (mol wt 51 kDa) has a specific activity of about 110 U/mg, nearly twice that (60 U/mg) of the intact 100-kDa enzyme, indicating that the kappa cat is virtually identical for both species. Unlike the parent enzyme, the C fragment is quite sensitive to inhibition by Pi (competitive vs ATP, noncompetitive vs Glc); sulfate and arsenate, but not acetate, inhibit with effectiveness similar to that seen with Pi. The Glc-6-P analog, 1,5-anhydroglucitol-6-P, also inhibits the C fragment (competitive vs ATP, uncompetitive vs Glc). Both N and C fragments bind to Affi-Gel Blue, an affinity matrix bearing a covalently attached analog of ATP, and are eluted by hexose 6-phosphates competitive with nucleotide binding to the parent enzyme. Based on the ability of various hexoses and hexose 6-phosphates (and analogs) to protect against guanidine-induced denaturation and subsequent proteolysis it is concluded that both fragments contain discrete sites for hexoses and hexose 6-phosphates, with specificities resembling those seen for the binding of these ligands to the parent enzyme. Synergistic interactions between the hexose and hexose-6-P binding sites, previously seen with the parent enzyme, are also observed with the C fragment but not the N fragment. The existence of binding sites for hexoses and hexose 6-phosphates on both halves conflicts with previous binding studies demonstrating a single hexose binding site and a single hexose 6-phosphate binding site on the intact 100-kDa enzyme, leading to the conclusion that one of each pair of sites must be latent in the intact enzyme, becoming manifest only in the isolated discrete halves. Several investigators have previously suggested that the 100-kDa mammalian hexokinases evolved by duplication and fusion of a gene encoding an ancestral 50-kDa Glc-6-P-insensitive hexokinase, similar to the present-day yeast enzyme, with sensitivity to Glc-6-P resulting from evolution of a duplicated catalytic site into a regulatory site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Functional characteristics of hexokinase bound to the type a and type B sites of bovine brain mitochondria.

Hexokinase is released from Type A sites of brain mitochondria in the presence of glucose 6-phosphate (Glc-6-P); enzyme bound to Type B sites remains bound. Hexokinase of freshly isolated bovine brain mitochondria (Type A:Type B, approximately 40:60) selectively uses intramitochondrial ATP as substrate and is relatively insensitive to the competitive (vs ATP) inhibitor and Glc-6-P analog, 1,5-anhydroglucitol 6-phosphate (1,5-AnG-6-P). After removal of hexokinase bound at Type A sites, the remaining enzyme, bound at Type B sites, does not show selectivity for intramitochondrial ATP and has increased sensitivity to 1,5-AnG-6-P. Thus, the properties of the enzyme bound at Type B sites are modified by removal of hexokinase bound at Type A sites. It is suggested that mechanisms for regulation of mitochondrial hexokinase activity, and thereby cerebral glycolytic metabolism, may depend on the ratio of Type A:Type B sites, which varies in different species.     

Enzymatic properties of overexpressed human hexokinase fragments

Full-length hexokinase (HK; ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), a truncate form of the enzyme lacking the first 11 amino acids (HK-11aa) and the 50 kDa C-terminal half (mini-HK) containing the catalytic domain, were overexpressed and purified to homogeneity to investigate the influence of the N-terminal region of human hexokinase type I (HK) on its regulatory properties. All forms of the enzyme are catalytically active with the HK-11aa being the most active. All the forms of HK showed the same affinity for glucose and MgATP and were also inhibited by glucose 6-phosphate (Glc 6-P) competitively vs. MgATP with similar Kis (28.5-37 M). Glucose 1,6-bisphosphate (Glc 1,6-P2) was also a strong inhibitor of all HKs without significant differences among the different truncate forms of the enzyme (Kis 49.5-59 M). At low concentrations (0-3 mM), Pi was able to reverse the sugar phosphate inhibition of the full-length HK and HK-11aa but not of the mini-HK. In contrast, at high concentrations Pi was an inhibitor of all the hexokinases investigated. These findings confirm that Pi has a low affinity binding site on the C-terminal of HK while counteracts glucose 6-phosphate inhibition by binding to or requiring the N-terminal half of the enzyme. The first 11 N-terminal amino acids influence the specific activity of HK but are unable to affect the kinetic properties investigated.     

Effect of ligand binding on the tryptic digestion pattern of rat brain hexokinase: relationship of ligand-induced conformational changes to catalytic and regulatory functions

The Type I isozyme of rat hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) is comprised of N- and C-terminal domains, associated with regulatory and catalytic functions, respectively. Extensive sequence similarity between the domains is consistent with evolution of the enzyme by gene duplication and fusion. Cleavage at tryptic sites located in the C-terminal domain is markedly sensitive to ligands present during digestion, while analogous sites in the N-terminal domain are either resistant to trypsin or unaffected by the presence of ligands. These results imply a lack of structural equivalence between the N- and C-terminal domains, with the overall structure of the N-terminal domain being "tighter" and with a major component of ligand-induced conformational changes being focused in the C-terminal domain. Based on a previously proposed structure for brain hexokinase, protection by substrate hexoses is attributed to substrate-induced closing of a cleft in the C-terminal domain. Similar protection at C-terminal cleavage sites results from binding of inhibitory hexose-6-phosphates to the N-terminal domain. In addition, hexose-6-phosphates evoke cleavage at a site, T5, located in a region that has been associated with binding of ATP to the C-terminal domain. Thus, alterations in this region, coupled with reduced accessibility resulting from cleft closure, may account for the mutually exclusive binding of inhibitory hexose-6-phosphates and substrate ATP. In the absence of Mg2+, all nucleoside triphosphates examined (ATP, UTP, CTP, and GTP) protected against digestion by trypsin. In contrast, ATP-Mg2+ stabilized the C-terminal domain but destabilized the N-terminal domain, while the chelated forms of the other nucleoside triphosphates were similar to the unchelated forms in their effect on proteolysis; the unique response to ATP-Mg2+ reflects the specificity for ATP as a substrate.     

A large-scale purification of recombinant histone H1.5 from Escherichia coli

The conversion of glucose into glucose 6-phosphate (Glc 6-P)1 traps glucose in a chemical state in which it cannot leave the cell and hence commits glucose to metabolism. In human tissues there are at least three hexokinase isoenzymes responsible for hexose phosphorylation. These enzymes are constituted by a single polypeptide chain with a molecular weight of approximately 100 kDa. Among these isoenzymes, hexokinase type I is the most widely expressed in mammalian tissues and shows reversion of Glc 6-P inhibition by physiological levels of inorganic phosphate. In this work the hexokinase I from human brain was overexpressed in Escherichia coli, as a hexahistidine-tagged protein with the tag extending the C-terminal end. An average of 900 U per liter of culture was obtained. The expressed protein was one-step purified by metal chelate affinity chromatography performed in NTA-agarose column charged with Ni(2+) ions. In order to stabilize the enzymatic activity 0.5 M ammonium sulfate was added to elution buffer. The specific activity of purified hexokinase I was 67.8 U/mg. The recombinant enzyme shows kinetic properties in agreement with those described for the native enzyme, and thus it can be used for biophysical and biochemical investigation.     

Homologous and Heterologous Interactions between Hexokinase and Mitochondrial Porin: Evolutionary Implications

Binding of the Type I isozyme of mammalian hexokinase to mitochondria is mediated by the porin present in the outer mitochondrial membrane. Type I hexokinase from rat brain is avidly bound by rat liver mitochondria while, under the same conditions, there is no significant binding to mitochondria from S. cerevisiae. Previously published work demonstrates the lack of significant interaction of yeast hexokinase with mitochondria from either liver or yeast. Thus, structural features required for the interaction of porin and hexokinase must have emerged during evolution of the mammalian forms of these proteins. If these structural features serve no functional role other than facilitating this interaction of hexokinase with mitochondria, it seems likely that they evolved in synchrony since operation of selective pressures on the hexokinase–mitochondrial interaction would require the simultaneous presence of hexokinase and porin capable of at least minimal interaction, and be responsive to changes in either partner that affected this interaction. Recent studies have indicated that a second type of binding site, which may or may not involve porin, is present on mammalian mitochondria. There are also reports of hexokinase binding to mitochondria in plant tissues, but the nature of the binding site remains undefined.     

Effect of ligand-induced conformational changes on the reactivity of specific sulfhydryl residues in rat brain hexokinase

Rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) contains 21 cysteine residues. On the basis of the amino acid sequence of the enzyme, these are predicted to be distributed among 14 peptides produced by tryptic digestion. Ten of these peptides, containing cysteine residues derivatized by reaction with the specific sulfhydryl reagent 2-bromoacetamido-4-nitrophenol have been identified in HPLC peptide maps; the four missing peptides are predicted to be relatively large and hydrophobic in character, properties that may have prevented their detection under the present conditions. The sequences encompassed by the 10 identified peptides include 12 of the 21 cysteine residues in the enzyme. The relative reactivity of these sulfhydryl groups with 2-bromoacetamido-4-nitrophenol has been assessed, and is in general accord with what might be predicted on the basis of their accessibility in the previously proposed structure for this enzyme. The effect of various ligands on reactivity of identified sulfhydryl groups has been determined; unique patterns of altered reactivity, resulting from ligand-induced conformational changes, have been observed. Biphasic effects were observed with increasing concentrations of either glucose 6-phosphate (Glc-6-P) or Pi. In both cases, decreased reactivity of sulfhydryls in the N-terminal half of the molecule was observed at low concentrations of the ligand, while further increase in ligand concentration resulted in decreased reactivity of sulfhydryl groups in the C-terminal half. In contrast, sulfhydryls in both N- and C-terminal halves were protected concomitantly by increasing concentrations of Glc. These results are consistent with previous studies that indicated (a) the existence of two sites for binding of Glc-6-P or Pi, a high affinity site in the N-terminal half and a site with lower affinity in the C-terminal half of the brain hexokinase molecule, and (b) binding of Glc to a single site located in the C-terminal half but evoking conformational effects throughout the molecule; the glucose analog, N-acetylglucosamine, previously shown to have more limited effects on conformation, affected reactivity of sulfhydryl groups only in the C-terminal half of the molecule. As reflected by effects on reactivity of sulfhydryl groups, conformational changes induced by binding of nucleotides depends markedly on the specific nature of the purine or pyrimidine base as well as the length and chelation status of the polyphosphate side chain. These results focus attention on specific regions of the molecule (the immediate environment of the sulfhydryl groups) that are affected by the binding of these ligands.     

Regulation of glucose metabolism in rat lung: Subcellular distribution,isozyme pattern,and kinetic properties of hexokinase

The subcellular distribution and isozyme pattern of hexokinase in rat lung were studied. Of the total hexokinase activity of lung, one-third was bound to mitochondria and one-third of the mitochondrial activity was in a latent form. The overt-bound mitochondrial hexokinase was specifically solubilized by physiological concentrations of glucose 6-phosphate and ATP. Inorganic phosphate partially prevented the solubilization by glucose 6-phosphate (Glc 6-P), whereas Mg²⁺ ions promoted rebinding of the solubilized enzyme to mitochondria. Thus, the distribution of hexokinase between soluble and particulate forms in vivo is expected to be controlled by the relative concentrations of Glc 6-P, ATP, P_i, and Mg²⁺. Study of the isozyme pattern showed that hexokinase types I, II, and III constitute the cell-sap enzyme of lung. The overt and latent hexokinase activities could be separately isolated by successive treatments of mitochondria with Glc 6-P and Triton X-100. The overt-bound activity consisted primarily of hexokinase type I, with a small proportion of type II isozyme. The latent activity, on the other hand, exclusively consisted of type I isozyme. Type I hexokinase, the predominant isozyme in lung, was strongly inhibited by intracellular concentration of Glc 6-P and this inhibition was counteracted by P_i. The bound form of hexokinase exhibited a significantly higher apparent K_i for Glc 6-P inhibition and a lower apparent K_m for ATP as compared to the soluble form. Thus, the particulate form of hexokinase is expected to promote glycolysis and may provide a mechanism for the high rate of aerobic glycolysis in lung.     

Rat brain hexokinase: location of the substrate hexose binding site in a structural domain at the C-terminus of the enzyme

A glucose analog, N-(bromoacetyl)-D-glucosamine (GlcNBrAc), previously used to label the glucose binding sites of rat muscle Type II and bovine brain Type I hexokinases, also inactivates rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) with pseudo-first-order kinetics. Inactivation occurs predominantly via a "specific" pathway involving formation of a complex between hexokinase and GlcNBrAc, but significant nonspecific (i.e., without prior complex formation) inactivation also occurs, and equations to describe this behavior are derived. Inactivation is dependent on deprotonation of a residue with an alkaline pKa, consistent with the modified residue being a sulfhydryl group as reported to be the case with the hexokinase of bovine brain. The affinity label modifies three residues (per molecule of enzyme) at indistinguishable rates, but only one of these residues appears to be critical for activity. Amino acid analysis of the modified enzyme indicates derivatization of three cysteine residues; there was no indication of modification of other residues potentially reactive with haloacetyl derivatives. Kinetic analysis and effects of protective ligands were consistent with location of the critical sulfhydryl at the glucose binding site. Peptide mapping techniques permitted localization of the critical residue, and thus the glucose binding site, in a 40-kDa domain at the C-terminus of the enzyme. This is the same domain recently shown to include the ATP binding site. Thus, catalytic function is assigned to the C-terminal domain of rat brain hexokinase.     

Association of the N- and C-terminal domains of phospholipase D. Contribution of the conserved HKD motifs to the interaction and the requirement of the association for Ser/Thr phosphorylation of the enzyme

Rat brain phospholipase D1 (rPLD1) belongs to a superfamily defined by the highly conserved catalytic motif (H(X)K(X)(4)D, denoted HKD. rPLD1 contains two HKD domains, located in the N- and C-terminal regions. The integrity of the two HKD domains is essential for enzymatic activity. Our previous studies showed that the N-terminal half of rPLD1 containing one HKD motif can associate with the C-terminal half containing the other HKD domain to reconstruct wild type PLD activity (Xie, Z., Ho, W.-T. and Exton, J. H. (1998) J. Biol. Chem. 273, 34679-34682). In the present study, we have shown by mutagenesis that conserved amino acids in the HKD domains are important for both the catalytic activity and the association between the two halves of rPLD1. Furthermore, we found that rPLD1 could be modified by Ser/Thr phosphorylation. The modification occurred at the N-terminal half of the enzyme, however, the association of the N-terminal domain with the C-terminal domain was required for the modification. The phosphorylation of the enzyme was not required for its catalytic activity or response to PKCalpha and small G proteins in vitro, although the phosphorylated form of rPLD1 was localized exclusively in the crude membrane fraction. In addition, we found that the individually expressed N- and C-terminal fragments did not interact when mixed in vitro and were unable to reconstruct PLD activity under these conditions. It is concluded that the association of the N- and C-terminal halves of rPLD1 requires their co-expression in vivo and depends on conserved residues in the HKD domains. The association is also required for Ser/Thr phosphorylation of the enzyme.     

Folding properties of functional domains of tropomodulin.

Tropomodulin (Tmod) stabilizes the actin-tropomyosin filament by capping the slow-growing end (P-end). The N- and C-terminal halves play distinct roles; the N-terminal half interacts with the N-terminal region of tropomyosin, whereas the C-terminal half interacts with actin. Our previous study (A. Kostyukova, K. Maeda, E. Yamauchi, I. Krieger, and Y. Maéda Y., 2000, Eur. J. Biochem. 267:6470-6475) suggested that the two halves are also structurally distinct from each other. We have now studied the folding properties of the two halves, by circular dichroism spectroscopy and by differential scanning calorimetry of the expressed chicken E-type tropomodulin and its large fragments. The results showed that the C-terminal half represents a single, independently folded unit that melts cooperatively through a two-state transition. In contrast, the N-terminal half lacks a definite tertiary structure in solution. The binding of N11, a fragment that corresponds to the first 91 amino acids of the tropomodulin, to tropomyosin substantially stabilized the tropomyosin. This may indicate that the flexible structure of the N-terminal half of tropomodulin in solution is required for binding to tropomyosin and that the N-terminal half acquires its tertiary structure upon binding to tropomyosin.