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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.

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.

Covalent interconversion of enzymes was first observed in glycogen phosphorylase from rabbit skeletal muscle (1–4). The interconversion involved the phosphorylation of the phosphorylase that converted the enzyme to its active form, and dephosphorylation returned the enzyme to its relatively inactive form. Since then, the number of enzymes/proteins reported to undergo covalent interconversion has mushroomed (5–7). Many of these interconvertible enzymes/proteins occupy key positions in metabolic networks, and their enzymic activities vary as a function of the fractional modification of the enzyme. Reversible covalent modifications can be grouped into seven classes based on the nature of the modification (Table 16.1). Of this list, the reversibility of tyrosine sulfation has yet to be established. Nevertheless, this process is likely to be reversible because protein sulfation has been reported to be involved in some essential regulatory pathways, e.g., tyrosine sulfation of interleukin-2 receptor (8), and reduced levels of tyrosine sulfate were found in retrovirus-induced transformed cells (9). Each of the modifications and de-modifications listed in Table 16.1 is catalyzed by specific converter enzymes; e.g., phosphorylation and dephosphorylation are catalyzed by protein kinases and phosphatases, respectively. This action of one enzyme on another constitutes a cascade, and the coupling of two opposing cascades forms a cyclic cascade system (Fig. 16.1).

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.

Publisher Summary This chapter discusses the cyclic cascade systems in metabolic regulation. The development of the cyclic cascade model in Earl Stadtman's laboratory is the result of detailed studies on the metabolic regulation of glutamine synthetase in Escherichia coli . Georges Cohen and Woolfolk demonstrated that glutamine synthetase is inhibited by eight end products of glutamine metabolism. The cyclic cascade model is derived from the dynamic coupling of a forward cascade and a reverse cascade. In its simplest form, a monocyclic cascade consists of a forward and a reverse converter enzyme and an interconvertible enzyme. The chapter presents the development of a simple in vitro model system that could be employed to investigate the validity of the theoretical predictions, and also presents the study of the bicyclic cascade of glutamine synthetase using both purified proteins and permeabilized cells. In essence, the cyclic cascade model developed in Earl Stadtman's laboratory reveals many advantageous reasons for biological systems to adopt this type of regulatory mechanism. Although the cyclic cascade model was developed mainly based on the detailed studies of glutamine synthetase, the model is applicable to all covalent interconvertible enzyme systems, such as those modified by phosphorylation, ADP-ribosylation, carboxymethylation, acetylation, and sulfation. This model may be applicable to the coupling of protein synthesis and degradation.