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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.

We report potent inhibition of the Mg(II).ATP-dependent protein phosphatase, Fc.M, by the regulatory subunit dimer of type II cAMP-dependent protein kinase, RII2. The protein kinase catalytic subunit has no effect on phosphatase activity and is unable to substitute for kinase FA in the kinase FA- and Mg(II).ATP-mediated phosphatase activation reaction. Phosphatase inhibition was investigated as a function of RII2 concentration. The results suggest that RII2 both inhibits the active phosphatase and inhibits phosphatase activation. The inhibition is shown to be noncompetitive with respect to substrate (phosphorylase a). The potential physiological significance of this inhibition is discussed in terms of phosphorylation/dephosphorylation cascade systems involving this kinase and phosphatase.

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.

Publisher Summary This chapter reviews the salient features of cyclic cascades with particular emphasis on phosphorylation/dephosphorylation systems. Phosphorylation/dephosphorylation is only one of many forms of reversible covalent modification of proteins and that the initial protein shown to be regulated by phosphorylation/dephosphorylation, glycogen phosphorylase, represents but a small fraction of all interconvertible enzymes and proteins. In fact, the entire spectrum of cellular activities is regulated by cyclic cascade systems, including hormonal and neurotransmitter responses, interferon action and susceptibility to viral infection, growth stimulation by polypeptide growth factors, viral cell transformation, chemical tumor promotion, DNA transcription and repair, protein synthesis, energy metabolism, and muscle contraction. Together with allosteric regulation and the control of enzyme levels through differential rates of protein synthesis and degradation, cyclic cascade systems maintain the living cell in a responsive, highly regulated state.