Kinetic Study of Yeast Hexokinase. 1. Steady-State Kinetics

Noat, Georges; Ricard, Jacques; Borel, Maguy; C, Got

doi:10.1111/j.1432-1033.1968.tb00337.x

Cited by 60 publications

(26 citation statements)

References 22 publications

(10 reference statements)

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“…The dissociation constant of MgATP from hexokinase in the absence of sugars is given by its K , in the slow ATPase reaction catalyzed by the enzyme. The values of 4-7 mM seen in the present work agree with those reported previously (Kaji & Colowick, 1965; Noat et al, 1969;DelaFuente et al, 1970), and the inhibition constant of MgADP as a competitive in- (Fromm & Zewe, 1962) while ATP analogues such as the one in which glucose replaces ribose (Hohnadel & Cooper, 1973) and ATP 6-glucose are uncompetitive (Danenberg & Danenberg, 1977). Lyxose also induces substrate inhibition by MgATP because of the synergistic binding between the two molecules (Danenberg & Cleland, 1975).…”

Section: Discussionsupporting

confidence: 95%

Substrate synergism and the kinetic mechanism of yeast hexokinase

Viola¹,

Raushel²,

Cleland³

1982

Biochemistry

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Michaelis constants for MgATP with yeast hexokinase vary from 28 microM with D-mannose to above 4 mM for the slow ATPase reaction, with the different values reflecting the degree of synergism in binding of MgATP and the sugar substrate. The best substrates show the greatest synergism, but the correlation is not exact. Similar synergistic binding between MgADP or its methylene analogue and phosphorylated sugars is seen. Product inhibiton of MgADP vs. MgATP and vice versa appears noncompetitive at low levels of variable substrate but becomes competitive at high levels. These patterns show that MgATP can combine with E-glucose-6-P (Ki = 4 mM) and MgADP with E-glucose (Ki = 1.6 mM). Isotope partitioning studies with glucose or glucose-6-P have determined the rates of release of these substrates from binary and ternary complexes and, together with reverse isotope exchange studies and the product inhibition studies mentioned above, have shown that the kinetic mechanism is a somewhat random one in which dissociation of sugars from productive ternary complexes is very slow, but release from nonproductive ternary complexes occurs at rates similar to those from binary enzyme-sugar complexes. D-Arabinose-5-P has a Km of 4.6 mM and a Vmax 5% that for glucose-6-P, confirming that the high Km for D-arabinose in the forward direction is caused by the low proportion in the furanose form. The dissociation constant of MgADP in the absence of sugars was determined from the Ki of 5.8 mM for MgADP as a competitive inhibitor vs. MgATP of the slow ATPase reaction.

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Section: Discussionsupporting

confidence: 95%

Substrate synergism and the kinetic mechanism of yeast hexokinase

Viola¹,

Raushel²,

Cleland³

1982

Biochemistry

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“…1A and B) and mutant cells (Fig. 1C and D) indicating an ordered bi-bi mechanism as described previously for hexokinase PII (21,29 (Table 3). Furthermore, hexokinase activity was greater than that in products of control matings with the untransformed recipient strain.…”

Section: Resultssupporting

confidence: 79%

Cloning of hexokinase structural genes from Saccharomyces cerevisiae mutants with regulatory mutations responsible for glucose repression.

Entian¹,

Hilberg²,

Opitz³

et al. 1985

Mol. Cell. Biol.

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The regulatory hexokinase PH mutants isolated previously (K.-D. Entian and K.-U. Frohlich, J. Bacteriol. 158:29-35, 1984) were characterized further. These mutants were defective in glucose repression. The mutation was thought to be in the hexokinase PII structural gene, but it did not affect the catalytic activity of the enzyme. Hence, a regulatory domain for glucose repression was postulated. For further understanding of this regulatory system, the mutationally altered hexokinase PII proteins were isolated from five mutants obtained independently and characterized by their catalytic constants and bisubstrate kinetics. None of these characteristics differed from those of the wild type, so the catalytic center of the mutant enzymes remained unchanged. The only noticeable difference observed was that the in vivo modified form of hexokinase PII, PIIM, which has been described recently (K.-D. Entian and E. Kopetzki, Eur. J. Biochem. 146:657-662, 1985), was absent from one of these mutants. It is possible that the PIIM modification is directly connected with the triggering of glucose repression. To establish with certainty that the mutation is located in the hexokinase PII structural gene, the genes of these mutants were isolated after transforming a hexokinaseless mutant strain and selecting for concomitant complementation of the nuclear function. Unlike hexokinase Pll wild-type transformants, glucose repression was not restored in the hexokinase Pll mutant transformants. In addition mating experiments with these transformants followed by tetrad analysis of sporulated diploids gave clear evidence of allelism to the hexokinase PlI structural gene.Glucose repression is the main regulatory system in the carbohydrate metabolism of Saccharomyces cerevisiae. In the presence of glucose in the medium the synthesis of many enzymes is repressed, particularly those involved in gluconeogenesis (18,19,36), the tricarboxylic acid cycle (30), glyoxylate cycle (1), and the catabolism of exogenously supplied sugars such as maltose (35), sucrose (20), and galactose (33). To elucidate the mechanism of glucose repression, mutants resistant to glucose repression have been isolated (39). Of the three independent mutant alleles hexi, hex2, and cat8O, hexi and hex2 showed changed hexokinase activities (14,16). Hexokinase activity was decreased in hex] mutants and was increased in hex2 mutants.Two native hexokinase isoenzymes, PI and PII have been described in S. cerevisiae (for a review see reference 6). A defect in the hexokinase PII structural gene has been shown to be responsible for decreased hexokinase activity in hexl mutants (9, 13), whereas hex2 has been shown to be a regulatory mutant allele which controls hexokinase PII synthesis, the synthesis of glucose-repressible enzymes, and maltose uptake (10, 11). The third gene involved in glucose repression (CA T80) had to be functional for the expression of increased hexokinase PII activity in hex2 mutants (15). Hence, hexokinase PII has a unique role in glucose repression, in which ...

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“…Hexokinase requires Mg 2+ for activity and G6PDH strongly prefers NADP + as the electron acceptor to oxidize G6P. Kinetic studies from several sources suggest that HK mediates phosphorylation of D-glucose via either the ordered Bi Bi mechanism (Noat et al 1968;Ricard et al 1972) or the random Bi Bi mechanism (Rudolph and Fromm 1970;Ganson and Fromm 1985). Magnesium ion is essential for the catalytic activities of HK, though it suppresses the fission yeast kinase activity at concentrations in excess of 10 mM presumably because of the formation of Mg 2 ATP (Noat et al 1970).…”

Section: Discussionmentioning

confidence: 96%

Purification and kinetic characterization of hexokinase and glucose-6-phosphate dehydrogenase fromSchizosaccharomyces pombe

Tsai

Chen²

1998

Biochem. Cell Biol.

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Hexokinase and D-glucose-6-phosphate dehydrogenase (G6PDH) from Schizosaccharomyces pmbe have been purified 250-fold by an identical three-step. Both enzymes are dimeric with a molecular mass of 88 kDa for the kinase and 112 kDa for the dehydrogenase. Steady-state kinetic studies were performed on hexokinase and G6PDH, which form the glucose phosphate branch of the oxidative pentose phosphate pathway of S. pombe (fission yeast). Hexokinase promotes Mg(2+)-activated phosphorylation of D-glucose by the equilibrium random Bi Bi mechanism with formation of the abortive enzyme-ADP-glucose complex. ADP inhibits the kinase competitively versus ATP and noncompetitively versus D-glucose. The Mg2+ activation of hexokinase is associated with an increase in the maximal velocity by its interaction with the ternary complex to facilitate the transfer of the phosphoryl group. G6PDH catalyzes NADP(+)-linked oxidation of D-glucose-6-phosphate by the ordered Bi Bi mechanism with NADP+ as the leading reactant. High NADP+ concentration inhibits the dehydrogenase by forming the dead-end ternary complex. In addition, G6PDH is also subjected to product inhibition by NADPH and noncompetitive inhibition by A(G)TP. Thus, the oxidative pentose phosphate pathway in S. pombe may be regulated via inhibition of hexokinase by ADP in conjunction with inhibition of G6PDH by NADPH and ATP.

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Kinetic Study of Yeast Hexokinase. 1. Steady-State Kinetics

Cited by 60 publications

References 22 publications

Substrate synergism and the kinetic mechanism of yeast hexokinase

Substrate synergism and the kinetic mechanism of yeast hexokinase

Cloning of hexokinase structural genes from Saccharomyces cerevisiae mutants with regulatory mutations responsible for glucose repression.

Purification and kinetic characterization of hexokinase and glucose-6-phosphate dehydrogenase fromSchizosaccharomyces pombe

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