Search

D-arabinose-5-triphosphate is a potent linear competitive inhibitor (K i = 60 μM) for substrates of E. coli RNA polymerase [E.C.2.7.7.6] in an in vitro transcription system employing AD111 DNA as template. In contrast the corresponding nucleotide triphosphate (5′ Ara-ATP) is without effect on the system. These facts coupled with other kinetic studies support the suggestion that the pentose triphosphate moiety of the normal substrate are bound at the catalytic site of the polymerase in the 3′ endo ribose conformation and require the 2′ hydroxyl in the axial position.

The mechanism of activation of the Mg(II) X ATP-dependent phosphatase by the kinase FA has been investigated. The inactive protein phosphatase can be represented as FC X M where FC is the inactive catalytic component and M is the heat-stable modulator protein (also known as inhibitor-2). Phosphorylation of the modulator protein is demonstrated during activation of FC X M. In addition, continuous ATP hydrolysis during the activation is observed. This suggests that a cyclic phosphorylation-dephosphorylation reaction is continuously occurring during the activation. It is proposed that phosphorylation of the modulator protein causes an isomerization in FC to generate an active phosphatase. The activated phosphatase is capable of dephosphorylating the phosphorylated modulator. Upon dephosphorylation of modulator, the active phosphatase returns to its inactive form via a slow isomerization.

A mechanism of activation of the ATP.Mg-dependent protein phosphatase (FC.M) has been proposed (Jurgensen, S., Shacter, E., Huang, C. Y., Chock, P. B., Yang, S.-D., Vandenheede, J. R., and Merlevede, W. (1984) J. Biol. Chem. 259, 5864-5870) in which a transient phosphorylation by the kinase FA of the modulator subunit (M) is the driving force for the transition of the inactive catalytic subunit (FC) into its active conformation. Incubation of FC.M with kinase FA and Mg2+ and adenosine 5'-(gamma-thio)triphosphate results in thiophosphorylation of M and also a conformational change in the phosphatase catalytic subunit; however, the enzyme remains inactive. Proteolysis of this inactive, thiophosphorylated complex causes proteolytic destruction of the modulator subunit and yields an active phosphorylase phosphatase species. Similar treatment of the native inactive enzyme does not yield active phosphatase. Evidence is presented, suggesting that a molecule of modulator is bound at an "inhibitory site" on the native enzyme. This modulator does not prevent the conformational change in the phosphatase catalytic subunit upon incubation with kinase FA and ATP.Mg but does partially inhibit the expression of the phosphorylase phosphatase activity.