RESUMO
Catalytic activity has been demonstrated for holotransketolase in the absence of free bivalent cations in the medium. The two active centers of the enzyme are equivalent in both the catalytic activity and the affinity for the substrates. In the presence of free Ca²âº (added to the medium from an external source), this equivalence is lost: negative cooperativity is induced on binding of either xylulose 5-phosphate (donor substrate) or ribose 5-phosphate (acceptor substrate), whereupon the catalytic conversion of the bound substrates causes the interaction between the centers to become positively cooperative. Moreover, the enzyme total activity increase is observed.
Assuntos
Cálcio/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcetolase/metabolismo , Cálcio/análise , Dextranos/química , Holoenzimas/química , Holoenzimas/isolamento & purificação , Holoenzimas/metabolismo , Cinética , Concentração Osmolar , Pentosefosfatos/metabolismo , Ribosemonofosfatos/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Tiamina Pirofosfato/análise , Tiamina Pirofosfato/metabolismo , Transcetolase/química , Transcetolase/isolamento & purificaçãoRESUMO
The effect of the type of the cation cofactor of transketolase (i.e., Ca2+ or Mg2+) on its interaction with xylulose 5-phosphate (donor substrate) has been studied. In the presence of magnesium, the active centers of the enzyme were functionally equivalent with respect to xylulose 5-phosphate binding and exhibited identical affinities for the donor substrate. Substitution of Ca2+ for Mg2+ results in the loss of the equivalence. In particular, this becomes apparent on binding of xylulose 5-phosphates to one of the two active centers of the enzyme, which caused the second center to undergo a several fold decrease in the affinity for the donor substrate.
Assuntos
Cálcio/farmacologia , Cátions Bivalentes/farmacologia , Magnésio/farmacologia , Pentosefosfatos/metabolismo , Transcetolase/metabolismo , Sítios de Ligação/efeitos dos fármacos , Cinética , Saccharomyces cerevisiae/enzimologia , Transcetolase/efeitos dos fármacosRESUMO
The interaction of transketolase with its acceptor substrate, ribose 5-phosphate, has been studied. The active centers of the enzyme were shown to be functionally nonequivalent with respect to ribose 5-phosphate binding. Under the conditions where only one out of the two active centers of transketolase is functional, their affinities for ribose 5-phosphate are identical. The phenomenon of nonequivalence becomes apparent when the substrate interacts with one of the two active centers. As a consequence of such interaction, the affinity of the second active center for ribose 5-phosphate decreases.
