Dimerization of brain hexokinase induced by its regulator glucose 6-phosphate

Bovine brain mitochondrial hexokinase type I, undergoes a concentration-dependent dimerization in presence of its product inhibitor glucose 6-phosphate. The effectiveness of this ligand in inducing the aggregation of brain hexokinase closely parallels its kinetic behavior as an inhibitor of this enzyme. ATP and inorganic phosphate known to antagonize the inhibitory effect of glucose 6-phosphate also cause a reversal of this dimerization process. ADP, another inhibitor of brain hexokinase, however, has no effect on the sedimentation behavior of the enzyme. It is suggested that the conformational alteration underlying the formation of hexokinase dimer in presence of glucose 6-phosphate has physiological significance

The effect of pH, temperature, and organic solvents on the kinetic parameters of Escherichia coli alkaline phosphatase

A study of the effect of pH on kinetic parameters of Escherichia coli alkaline phosphatase, in presence and in absence of organic solvents, has shown that a cationic acid group with a pK of 7.4 at 25.5° is implicated in catalysis and that a neutral acid group with a pK of 9.2 at 25.5° is involved in enzyme-substrate binding. From the change in pK with temperature, values of 6,500 and 11,000 cal per mole, respectively, have been calculated for the heats of ionization of these two groups. These data are consistent with the proposal that the imidazolium group of a histidine residue participates in catalysis, and that a water molecule coordinated to the zinc atom functions in enzyme-substrate binding

The histidyl residues in ribonuclease-S. Photooxidation in solution and in single crystals; the iodination of histidine-12

With molar ratios of methylene blue to ribonuclease-S of 1:30, histidyl residues 105 and 119 are destroyed by photooxidation at approximately equal rates. The loss of histidine 119 results in complete loss of enzymic activity; the loss of 105 is without effect on the activity. Histidine-12 in RNase-S is photooxidized at a slower rate than 105 or 119 and slower than the same residue in S-Peptide. Apparently histidine-12 is partially protected in the RNase-S complex. Loss of histidine-12 in S-Peptide results in loss of potential activity. Photooxidation of S-Peptide causes the oxidation of methionine-13 to the sulfoxide as well as the destruction of histidine-12. The sulfoxide can easily be reduced to yield a derivative where the only change is in histidine-12. Monoiodination of histidine-12 in S-Peptide on carbon-2(4) also resulted in complete loss of potential activity. Estimates of ratio of S-Protein to S-Peptide derivative association constants relative to that for the unmodified peptide show reductions in affinity by factors of about 700 for methionine-13 sulfone, 1900 for photooxidized histidine-12, 3200 for 2(4)-iodohistidine-12. The loss of histidine-119 markedly lowers the affinity of S-Protein for native S-Peptide. The photooxidation reaction was carried out in the solid state on single crystals of RNase-S. The qualitative effects were identical with those seen in solution, although the overall rate was slower. A comparison of X-ray diffraction patterns of native and photooxidized crystals of RNase-S showed only slight intensity changes. There was no evidence of any disordering of the crystal lattice. It is tentatively concluded that only small general conformational changes can have occurred, and that the activity effects can probably be attributed directly to the loss of the imidazole residues

Bovine brain mitochondrial hexokinase: solubilization, purification, and role of sulfhydryl residues

Bovine brain mitochondrial hexokinase, type I, has been solubilized by extraction of the mitochondria in 0.2 m acetate buffer, pH 5.0, containing 0.9 m NaCl. The solubilized enzyme has been purified to apparent homogeneity as shown by ultracentrifugal and electrophoretic criteria. The purification procedure included fractionation of the solubilized enzyme with ammonium sulfate and two successive diethylaminoethyl cellulose chromatographic steps. The sedimentation coefficient, S20,w, was found to be 5.9 S at a protein concentration of 1.7 mg per ml. The approximate molecular weight as determined by gel filtration on Sephadex G-200 is 107,000. The enzyme has 11 to 13 sulfhydryl residues per mole as determined by reaction of the denatured enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Almost all of these residues react with DTNB in the native enzyme though with differing degrees of reactivity. Reaction of the enzyme with excess DTNB caused its rapid inactivation. A comparison of the progress of this inactivation with the progress of the reaction of the sulfhydryl residues of the enzyme with DTNB showed that a maximum of only 2 residues could be involved in the inactivation process. If 2-mercaptoethanol is added to the enzyme immediately after complete inactivation, a rapid and total recovery of enzyme activity ensues. These results have been analyzed in terms of involvement of sulfhydryl residues, in the active conformation of the enzyme. Substrate glucose partially protects the enzyme against inactivation by DTNB and also modifies the reactivity of the sulfhydryl residues of the enzyme toward this reagent. MgATP, MgADP, and inorganic phosphate even at 10 mm concentration do not protect the enzyme against inactivation by DTNB. Product inhibitor glucose 6-phosphate affords a complete protection to the enzyme against inactivation by DTNB and drastically changes the reactivity of its sulfhydryl residues. Fructose 6-phosphate is without a comparable effect

Magnetic resonance studies on the interaction of metal-ion and nucleotide ligands with brain hexokinase

Our previous studies have shown that one manganous ion binds tightly to bovine brain hexokinase, with a Kd =25 ± 4 μM. The characteristic proton relaxation rate (PRR) enhancement of this binary complex (εb) 3 .5 at 9 MHz and 23 °C [ Jarori, G. K. Kasturi, S. R., and Kenkare, U. W. (1981) Arch. Biochem. Biophys. 211,258 - 2681. On the basis of PRR enhancement patterns, observed on the addition of nucleotides ATP and ADP to this E . Mn binary complex, we now show the formation of a nucleotide-bridge ternary complex, enzyme . nucleotide . Mn. Addition of glucose 6-phosphate to enzyme . ATP . Mn, results in a competitive displacement of ATP Mn from the enzyme. However, a quaternary complex E · ADP· Mn· Glc-6-P appears to be formed when both the products are present. β, γ-Bidentate Cr(II1)ATP has been used to elucidate the role of direct binding of Mn(I1) in catalysis, and the stoichiometry of metal-ion interaction with the enzyme in the presence of nucleotide. Bidentate Cr(II1)ATP serves as a substrate for brain hexokinase without any additional requirement for a divalent cation. However, electron-spin resonance studies on the binding of Mn(I1) to the enzyme in the presence of Cr(I1I)ATP suggest that, in the presence of nucleotide, two metal ions interact with hexokinase, one binding directly to the enzyme and the second interacting via the nucleotide bridge. It is this latter one which participates in catalysis. Experiments carried out with hexokinase spin-labeled with 3-(2-iodo-acetamido)-2,2,5,5-tetramethyl-lpyrrolidinyloxyl clearly showed that the direct-binding Mn site on the enzyme is distinctly located from its ATP Mn binding site

Reaction of brain hexokinase with tetranitromethane: oxidation of essential thiol groups

Treatment of bovine brain mitochondrial hexokinase with a fivefold molar excess of tetranitromethane (TNM) at pH 8.0 results in a complete loss of activity of the enzyme. Spectral measurements and amino acid analysis showed that only cysteinyl residues were modified by TNM under these conditions. The products of the reaction of TNM with the enzyme were cysteic acid and disulfides as found by amino acid analysis of the carboxymethylated hexokinase. Failure of 2-mercaptoethanol to reverse the effect of TNM suggested that the thiol group that is oxidized to cysteic acid is the one essential for enzyme activity. Substrates glucose and ATP, inhibitors ADP and glucose 6-phosphate, and the effector Pi partially protect against the inactivation of the enzyme by TNM. Studies with the carboxymethylated and performic acid-oxidized enzyme showed that it contained disulfide bonds when stored in the absence of 2-mercaptoethanol. The total number of half-cystines in hexokinase, present as cysteines and disulfides, is estimated to be about 20. A study of the reaction of TNM with the inactive enzyme derivative, prepared by reaction with stoichiometric amounts of 5,5'-dithiobis(2-nitrobenzoic acid) [Redkar, V. D., and Kenkare, U. W. (1975), Biochemistry, 14, 4704], indicated that one more thiol, thus far not implicated in enzyme function, is important for the active conformation of the enzyme

Inactivation of brain hexokinase by an adenosine 5'-triphosphate analog

An analog of ATP, 6-mercapto-9-β-D -ribofuranosylpurine 5'-triphosphate (SH-TP), functions as a phosphoryl donor for the reaction catalyzed by hexokinase. Incubation of the enzyme with millimolar concentrations of this reagent led to its rapid inactivation. However, the corresponding monophosphate derivative of this reagent was much less effective. A plot of the initial rate of inactivation versus the concentration of SH-TP exhibited saturation, suggesting the formation of a reversible complex between the enzyme and SH-TP prior to the inactivation reaction. The dissociation constant of this enzyme-inhibitor complex was the same as the Km of this reagent with respect to hexokinase in the phosphoryl transfer reaction. These data and protection by the substrates glucose, ATP, and ITP against this inactivation indicate that SH-TP is an active site-directed reagent for hexokinase. The reactivation of the inactivated enzyme by reducing thiol reagents and the disappearance of free thiols after reaction with SH-TP show that sulfhydryl groups of the enzyme are those modified. However, no enzyme bound SH-TP could be demonstrated. Progress curves of inactivation of the enzyme by SH-TP, plotted in a first-order fashion, showed biphasicity. These results are explained by a proposal that two thiols, one essential and the other nonessential, lie in close proximity at the active site of hexokinase

Insulin-like growth factor I induces tumor hexokinase RNA expression in cancer cells

Increased glycolysis is a characteristic of cancer cells. Though less efficient in energy production, it ensures continuous supply of energy and phosphometabolites for biosynthesis enabling metastatic and less vascularized cancer cells to proliferate even under hypoxic conditions. Since hexokinase is the first rate limiting enzyme in the glycolytic pathway, elevated levels of Type II like hexokinase in tumors are of great significance in this context. Under normal conditions insulin regulates expression of hexokinase Type II isoenzyme, which is predominantly expressed in muscle. On the other hand cancer cells overexpress insulin-like growth factors and their receptors which mimic many activities of insulin. This prompted us to examine a hypothesis that insulin-like growth factors may be responsible for overexpression of tumor hexokinase. Our experiments demonstrate that insulin-like growth factor I indeed induces hexokinase gene expression in a concentration and time dependent manner in two cancer cell lines we studied

Expression of two type II-like tumor hexokinase RNA transcripts in cancer cell lines

To maintain an elevated glycolytic rate, cancerous or proliferating cells alter the expression pattern of rate limiting glycolytic enzymes. Since glucose phosphorylation is the first step in glycolysis, hexokinase (HK), the first rate limiting glycolytic enzyme, can play a key regulatory role in this process. A low-Km, mitochondrial type II-like tumor HK is described as the predominant form in hepatomas. However, recent identification of a high-Km glucose phosphorylating activity in a range of cancer cells prompted us to characterize glucose phosphorylating enzymes of cancer cells at the molecular level. Highly sensitive reverse-transcription polymerase chain reaction identifies an induction and overexpression of a type II-like tumor HK RNA in a range of cancer cell lines irrespective of tissue origin. In addition, we report here the identification of two RNA transcripts of type II-like tumor HK of ~5.5 and ~4.0 kb in these cancer cells lines, including muscle-derived L6 myoblast cells. Interestingly, under normal conditions muscle cells express only a ~5.5-kb type II HK RNA transcript. A significant amount of type I HK RNA was also found expressed in cancer cell lines. RNA encoding glucokinase (GK), the high-Km HK isozyme, was found only in cancer cells originating from liver and pancreas, which express GK under normal conditions

Reaction of brain hexokinase with a substrate-like reagent. Alkylation of a single thiol at the active site

An analogue of the substrate glucose, N-(bromoacetyl)-D-glucosamine (GlcNBrAc) inactivates bovine brain mitochondrial hexokinase completely and irreversibly in a pseudo-first-order fashion at pH 8.5 and 22°C. The rate of inactivation of hexokinase by this reagent does not increase linearly with increasing reagent concentration but exhibits an apparent saturation effect, suggesting the formation of a reversible complex between the enzyme and the reagent prior to the inactivation step. The pH dependence of the rate of inactivation suggests that a group on the enzyme with pKa = 9.1 is being modified by this reagent. At pH 8.0 the rate of inactivation by this reagent is very slow, and it can be shown to be a competitive inhibitor of the hexokinase reaction with respect to the substrate glucose. The substrates glucose and ATP strongly protected the enzyme against the inactivation reaction. The inactivation of the enzyme was found to be accompanied by the alkylation of two sulfhydryl residues as shown by the formation of approximately 2 mol of S-(carboxymethyl)-cysteine/mol of inactivated enzyme. Treatment of the enzyme with 14C-labeled reagent results in the incorporation of approximately 2 mol of reagent/mol of inactivated enzyme. However, the enzyme protected by glucose still shows the incorporation of approximately 1 mol of the labeled reagent/mol of the enzyme. From a tryptic digest of the enzyme inactivated by this reagent, two labeled peptides were obtained, one of which was absent if the labeling reaction was carried out in presence of glucose. These results indicate that the affinity reagent reacts with two thiols, only one of which is crucial for the activity of the enzyme and is located in the region of its active site