Enzymes phosphorylation

Family of enzymes phosphorylating phosphatidylinositol (Ptdlns), PtdIns(4)phosphate, and PtdIns(4,5)phosphate in the 3-position. The Ptdlns(3 phospholipids are second messengers in processes like cell growth, cytoskeletal rearrangement, and vesicular transport. PI 3-kinases are heterodimers composed of a catalytic and a regulatory subunit. The enzymes are activated by insulin, many growth factors, and by a variety of cytokines. Their activity can be inhibited by wortmannin and LY294002. 

The general picture of muscle contraction in the heart resembles that of skeletal muscle. Cardiac muscle, like skeletal muscle, is striated and uses the actin-myosin-tropomyosin-troponin system described above. Unlike skeletal muscle, cardiac muscle exhibits intrinsic rhyth-micity, and individual myocytes communicate with each other because of its syncytial nature. The T tubular system is more developed in cardiac muscle, whereas the sarcoplasmic reticulum is less extensive and consequently the intracellular supply of Ca for contraction is less. Cardiac muscle thus relies on extracellular Ca for contraction if isolated cardiac muscle is deprived of Ca, it ceases to beat within approximately 1 minute, whereas skeletal muscle can continue to contract without an extraceUular source of Ca +. Cyclic AMP plays a more prominent role in cardiac than in skeletal muscle. It modulates intracellular levels of Ca through the activation of protein kinases these enzymes phosphorylate various transport proteins in the sarcolemma and sarcoplasmic reticulum and also in the troponin-tropomyosin regulatory complex, affecting intracellular levels of Ca or responses to it. There is a rough correlation between the phosphorylation of Tpl and the increased contraction of cardiac muscle induced by catecholamines. This may account for the inotropic effects (increased contractility) of P-adrenergic compounds on the heart. Some differences among skeletal, cardiac, and smooth muscle are summarized in... 

So far, it has been established from in vitro studies that the enzyme undergoes phosphorylation, a process that changes the conformation of the enzyme protein and leads to an increase in its activity. This involves Ca +/calmodulin-dependent protein kinase II and cAMP-dependent protein kinase which suggests a role for both intracellular Ca + and enzyme phosphorylation in the activation of tryptophan hydroxylase. Indeed, enzyme purified from brain tissue innervated by rostrally projecting 5-HT neurons, that have been stimulated previously in vivo, has a higher activity than that derived from unstimulated tissue but this increase rests on the presence of Ca + in the incubation medium. Also, when incubated under conditions which are appropriate for phosphorylation, the of tryptophan hydroxylase for its co-factor and substrate is reduced whereas its Fmax is increased unless the enzyme is purified from neurons that have been stimulated in vivo, suggesting that the neuronal depolarisation in vivo has already caused phosphorylation of the enzyme. This is supported by evidence that the enzyme activation caused by neuronal depolarisation is blocked by a Ca +/calmodulin protein kinase inhibitor. However, whereas depolarisation... 

This can be used in several ways. H+-ATPase plays a key role in oxidative phosphorylation (ATP synthesis) as it transfers protons back into the matrix space with simultaneous synthesis of ATP (with temporary enzyme phosphorylation, cf. page 451). 

Figure 11.8). In addition, the activated enzyme phosphorylates itself, and thus remains partly active even after the Ca2+ concentration falls and calmodulin is released from the enzyme. In contrast to the CaM kinases, another important target of Ca2+-cahnodulin is the plasma membrane Ca2+-ATPase pump, whose activation drives down the Ca2+ concentration within the cell, helping to terminate the signal. 

Although the 5/6-kinase was not detected in the final preparation of the CSN from human erythrocytes [31], it cannot be excluded that the en2yme is associated with another pool of CSN particles. The enzyme phosphorylates c-Jun, IkB as well as p53 and is sensitive to curcumin. These characteristics are very similar to those described for CK2 and PKD. It has been shown that the 5/6-kinase interacts with CSNl and that over-expression of CSNl inhibits its activity [24]. It might be that it interacts with the N-terminal part of CSNl, which has been shown to suppress activation of an AP-1 promoter [22]. Future studies will show whether additional kinases besides 5/6-kinase, CK2 and PKD can interact with the CSN under certain circumstances. For example, an interaction of CSN3 with IKKy, a component of the IKK kinase complex, has been published [32]. 

Rashba-Step, J., Tatoyan, A., Duncan, R,. Ann, D., Pushpa-Rehka, T.R., and Sevanian, A, 1997, PhosphoUpid peroxidation induces cytosolic phospholipase A2 activity membrane effects versus enzyme phosphorylation, Arcii. Biochem. Biophys. 343 44-54. 

This enzyme phosphorylates HMWj (or MAP ) >n a manner which affects the associadon of acdn and microtubule networks (Sloboda et al., 1975 Pollard et al., 1982)... 

Protein kinase (stimulated by calcium ions) This enzyme phosphorylates tubulin in a calcium ion-acdvated process (Burke and De-... 

Lysosomal enzymes phosphorylation of mannose by phosphotransferase in Golgi I-cell disease... 

Histone kinases responsible for N-phosphorylation have been isolated from regenerating rat liver [109] and Walker-256 carcinosarcoma cells [110]. One kinase with a pH optimum of 9.5 phosphorylated His-18 and His-75 of H4, while the other with a pH optimum of 6.5 phosphorylated lysine of HI. The enzyme from regenerating rat liver phosphorylated H4 at 1-phosphoryl histidine, while the carcinosarcoma enzyme phosphorylated H4 His at the position 3 [111]. Both kinases were cAMP independent [110]. Matthews and colleagues purified a 32-kDa histidine H4 kinase from yeast, Saccharomyces cerevisiae [112,113]. The enzyme phosphorylated His-75 (1-phosphoryl histidine) in H4. His-18 of H4 and other histidines in other core histones were not phosphorylated by this kinase [112]. Protein phosphatases 1, 2A, and 2C could dephosphorylate His-75 of H4 [114]. Applying a gel kinase approach to detect mammalian H4 histidine kinases, Besant and Attwood detected four activities in the 34-41 kDa range with extracts from porcine thymus [115]. 

This enzyme phosphorylates branched-chain a-keto acid dehydrogenase using ATP. 

In both schemes, the specificities of the pump for catalysis change in the two enzyme states. Jencks points out that coupling is determined (a) by the chemical specificity achieved in catalyzing phosphoryl transfer to and from the enzyme (wherein E-Ca2 reversibly binds ATP, and E reacts reversibly with orthophosphate), and (b) by the vectorial specificity for ion binding and dissociation (wherein E reversibly binds/dissociates cytoplasmic calcium ion, and E—P reversibly binds/dissociates luminal calcium). There must be a single conformation change during the reaction cycle between Ei and E2 in the free enzyme and from Ei P-Ca2 to E2-P-Ca2 after enzyme phosphorylation. 

Important reactions of intermediary metabolism regulated by enzyme phosphorylation. Key Blue text = intermediates of carbohydrate metabolism ... 

Cardini, C. E. and Leloir, L. F. 1952. Enzymic phosphorylation of galactosamine and galactose. Arch. Biochem. Biopkys. 45, 55-64. 

Ester + enzyme phosphoryl enzyme + alcohol (leaving group) U II... 

The authors experience is that the enzymic phosphorylation and diphosphorylation of nucleoside monophosphates is very efficient the yields are nearly quantitative and the immobilized enzyme system appears reusable for at least three months. 

This section emphasizes work done in the last few years. The reader is referred to other sources for reviews of older work236 or more general discussions of nucleophilic reactions at phosphorus.237"245 More general discussions of enzymic phosphoryl and nucleotidyl transfer are available,246 248 and the role of divalent metal ions has been reviewed.249"251... 

Enzymes are sometimes inactive as simple polypeptides. Addition of another group forms the fully active enzyme. Phosphorylation or glycosylation of an OH or NH in an R-group is typical. Phosphorylations are especially common, and this process is itself mediated by another enzyme, called a kinase. Kinases are an important family of enzymes, and kinase inhibition can be a powerful means of impacting cellular function. 

Depending on the specific organophosphate, some phosphorylated enzyme may be reactivated ("dephosphorylated") by certain oxime antidotes from one to two days after OP absorption. Thereafter a change in the nature of the enzyme-phosphoryl bond occurs, rendering the inactivation irreversible and necessitating the generation of new enzyme. 

Simple additions of such labile aquo ions as Mg, Ca, Zn and Mn "1", which are of importance in enzymic phosphoryl transfer, have resulted in only very modest catalytic effects for reactions of phosphate species (3). The much more effective t Com unit possesses the special advantage that it remains intact for long periods, while trans/cis isomerization and substitution in the fifth and sixth coordination sites proceed at moderately rapid and generally convenient rates. These characteristics make it particularly suitable for use in model studies (4). 

In liver, the cells contain mainly glucokinase instead of hexokinase and this enzyme phosphorylates only glucose. Thus in liver, fructose is metabolized instead by the fructose 1-phosphate pathway (Fig. 2). 

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