Effect of ligands on the reactivity of essential sulfhydryls in brain hexokinase. Possible interaction between substrate binding sites

Redkar, Vilas D.; Kenkare, Umakant W.

doi:10.1021/bi00692a022

Cited by 35 publications

(9 citation statements)

References 27 publications

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“…Though the underlying mechanism for the difference between the two groups has not yet been clarified, the positional relation between the inhibitor site and substrate site on the isoenzyme molecule might be different between the two groups. Thus the present results may be related to those reported by Redkar & Kenkare (1972) for brain mitochondrial hexokinase. They described that glucose was protective against the inactivating action of 5,5'-dithiobis-(2-nitrobenzoic acid), another thiol inhibitor.…”

Section: Resultssupporting

confidence: 92%

Difference in kinetic properties between hexokinase type I isoenzymes from various rat tissues with reference to the effect of a thiol inhibitor (Short Communication)

Kamikashi

Kizaki

Murakami

et al. 1974

Biochemical Journal

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Section: Resultssupporting

confidence: 92%

Difference in kinetic properties between hexokinase type I isoenzymes from various rat tissues with reference to the effect of a thiol inhibitor (Short Communication)

Kamikashi

Kizaki

Murakami

et al. 1974

Biochemical Journal

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“…A kinetic model, however, can also account for a limiting release of HKI: some fraction of the total bound HKI could be "stuck" on the membrane, because of an extremely slow step in a distinct release mechanism. The kinetic model is unlikely because ATP rapidly releases 90% of bound hexokinase from ascites mitochondria (6), and various salts also release nearly all bound HKI (6,56,57). Hence, the limiting release of ϳ60% induced by Glu-6-P is most likely an equilibrium phenomenon.…”

Section: Discussionmentioning

confidence: 99%

Glucose 6-Phosphate Release of Wild-type and Mutant Human Brain Hexokinases from Mitochondria

Skaff

Kim

Tsai

et al. 2005

Journal of Biological Chemistry

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One molecule of glucose 6-phosphate inhibits brain hexokinase (HKI) with high affinity by binding to either one of two sites located in distinct halves of the enzyme. In addition to potent inhibition, glucose 6-phosphate releases HKI from the outer leaflet of mitochondria; however, the site of glucose 6-phosphate association responsible for the release of HKI is unclear. The incorporation of a C-terminal polyhistidine tag on HKI facilitates the rapid purification of recombinant enzyme from Escherichia coli. The tagged construct has N-formyl methionine as its first residue and has mitochondrial association properties comparable with native brain hexokinases. Release of wild-type and mutant hexokinases from mitochondria by glucose 6-phosphate follow equilibrium models, which explain the release phenomenon as the repartitioning of ligand-bound HKI between solution and the membrane. Mutations that block the binding of glucose 6-phosphate to the C-terminal half of HKI have little or no effect on the glucose 6-phosphate release. In contrast, mutations that block glucose 6-phosphate binding to the N-terminal half require ϳ7-fold higher concentrations of glucose 6-phosphate for the release of HKI. Results here implicate a primary role for the glucose 6-phosphate binding site at the N-terminal half of HKI in the release mechanism.Brain tissue relies almost exclusively on glucose as its source of energy. Approximately 25% of circulating blood glucose and 20% of consumed oxygen is utilized by the brain, an organ that constitutes less than 2% of the human body mass (1). Although four isozymes of hexokinase (ATP:D-hexose 6-phosphotransferase (2.7

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“…The results of the purification are summarised in Table 1. The specific activity of the wheatgerm LII enzyme must be contrasted with the much higher values of the yeast and mammalian enzymes [12,13,16,17,19,20].…”

Section: Purificationmentioning

confidence: 98%

Wheatgerm Hexokinase: Physical and Active‐Site Properties

Higgins

Easterby

1974

European Journal of Biochemistry

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The LII form of wheatgerm hexokinase has been purified and is homogeneous by criteria of electrophoresis and ultracentrifugation. The purified enzyme has a specific activity of 3.5 units/mg with respect to glucose and is a single polypeptide chain of 51000 molecular weight.On the basis of K, values, fructose appears to be the preferred hexose substrate in contrast to the hexokinases of mammals and yeast. Data from "dead-end" inhibition studies suggest that a rapid-equilibrium random mechanism obtains but end-product inhibition indicates that the mechanism may show some additional complexities.The pH dependence of the alkylation and inactivation of the enzyme by iodoacetamide indicates the participation of a group of pKB 6.58 in these processes. This group is not itself alkylated, but its state of protonation determines the rate of the alkylation reaction. Substrates and products affect the rate of alkylation, and the dissociation constants of the enzyme -substrate and enzyme -product complexes are similar to the K m and Ki values for these ligands. These data also are consistent with a random mechanism. Studies on the pH dependence of photo-oxidation of the enzyme show a bisphasic loss of enzyme activity with respect t o time. Two ionizing groups with pKa values of 6.78 and 7.44 are involved in the first and second phases, respectively. Investigation of the effects of ligands on the rate of photooxidation shows that, for the first phase, the values for the dissociation constants are equal to the Km and Ki values for the substrates and products.However, for the second phase the values are greatly elevated. This may imply that a conformation change occurs in the enzyme following the initial phase of photo-oxidation.A possible role for hexokinases in germinating cereal seeds is discussed.Hexose 6-phosphates, particularly glucose 6-phosphate, provide a focal point for many of the pathways of carbohydrate metabolism. Hexokinase is responsible for initiating the metabolism of glucose and other hexoses within the cell and is, therefore, a. potential site of regulation of hexose metabolism.Hexokinase activity has been detected in several plant tissues [1-111. Latzko and KotzB [I] have measured steady-state metabolite concentrations, in

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Effect of ligands on the reactivity of essential sulfhydryls in brain hexokinase. Possible interaction between substrate binding sites

Cited by 35 publications

References 27 publications

Difference in kinetic properties between hexokinase type I isoenzymes from various rat tissues with reference to the effect of a thiol inhibitor (Short Communication)

Difference in kinetic properties between hexokinase type I isoenzymes from various rat tissues with reference to the effect of a thiol inhibitor (Short Communication)

Glucose 6-Phosphate Release of Wild-type and Mutant Human Brain Hexokinases from Mitochondria

Wheatgerm Hexokinase: Physical and Active‐Site Properties

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