Kinase and phosphatase enzymes

Control of pymvate dehydrogenase activity is via covalent modification a specific kinase causes inactivation of the PDH by phosphorylation of three serine residues located in the pyruvate decarboxylase/dehydrogenase component whilst a phosphatase activates PDH by removing the phosphates. The kinase and phosphatase enzymes are non-covalently associated with the transacetylase unit of the complex. Here again we have an example of simultaneous but opposite control of enzyme activity, that is, reciprocal regulation. 

Mammalian sperm contain tyrosine kinase activities (Berruti and Martegani, 1989). In addition, the tyrosine phosphorylation of a limited array of sperm proteins is increased during capacitation (Leyton and Saling, 1989 Duncan and Fraser, 1993 Visconti et al., 1995a,b Morte et al., 1998). In general, the relevant kinase and phosphatase enzymes have not yet been identified (Morte et al., 1998). 

The different effects of glucagon and epinephrine in the liver and in muscle are due to the different properties of the kinase and phosphatase enzymes that catalyze the synthesis and degradation of fructose 2,6-bisphosphate in these organs. In the liver, the... 

Acetyl-CoA carboxylase activity can be altered by interaction with citrate and other tricarboxylic acids. Much attention has been paid to hepatocyte systems where compounds such as glucagon or dibutyryl cyclic AMP lower cytosolic citrate levels which cause a lowered acetyl-CoA carboxylase activity (Lane et al, 1979). The latter is also inhibited by raised fatty acyl-CoA concentrations which cause depolymerization of the mammalian carboxylase. Information is available concerning inhibitor specificity and interactions with other subcellular compounds (cf. Wakil et al, 1983). The mammalian acetyl-CoA carboxylase has also been reported to undergo covalent modification through phosphorylation/ dephosphorylation. Such regulation may involve the simultaneous presence of Co A (Yeh et aL, 1981). Various kinase and phosphatase enzymes which may be important in the modification of the mammalian acetyl-CoA carboxylase have been purified (cf. Wakil etal., 1983). 

Phosphorylation by protein kinases of specific seryl, threonyl, or tyrosyl residues—and subsequent dephosphorylation by protein phosphatases—regulates the activity of many human enzymes. The protein kinases and phosphatases that participate in regulatory cascades which respond to hormonal or second messenger signals constimte a bio-organic computer that can process and integrate complex environmental information to produce an appropriate and comprehensive cellular response. 

It is worth mentioning that membrane-bound forms of GC, which can be considered signal transducing enzymes , are structurally homologous to other signal transducing enzymes, such as certain protein tyrosine kinases and phosphatases, which also possess receptor moieties in their extracellular (amino terminus) domain and enzyme catalytic activity in their intracellular domain (see Ch. 24). Activation of many of these receptors occurs upon ligand-induced dimerization of the receptors, and a similar... 

The switch in the action of the enzyme between its kinase and phosphatase activities is brought about by phosphorylation mediated by the serine/threonine protein kinase A (PKA), the same cAMP dependent enzyme which plays a role in the control of glycogen metabolism. In its kinase form, PFK-2 is dephosphorylated but phosphorylated in the phosphatase form. 

Phosphorylation, like most biological processes, is dynamic. The enzymes responsible for the removal of phosphate moieties are known as phosphatases. There are remarkably fewer phosphatases than kinases (about half), and unsurprisingly phosphatases are less specific than kinases. The dynamic nature of phosphorylation comes into effect when considering signal transduction in vivo. Kinases and phosphatases are complementary to each other, where one enzyme can turn a signal on and the complementary enzyme will turn the signal off. 

Activity is modulated by other proteins present in the membrane. These include a glycoprotein (MW 53 000) which stimulates ATPase activity 138 a 60 000 molecular weight protein, which is phosphorylated in a calmodulin-dependent fashion, affects accumulation of calcium 139 while the activity of the enzyme is affected by an endogenous kinase and phosphatase which phosphorylates and dephosphorylates the protein.140 Phospholamban is a proteolipid (MW 22 000) in cardiac SR which undergoes both cyclic AMP-dependent and calcium-calmodulin-dependent phosphorylation,141 but at different sites. All these proteins are probably involved in regulating the activity of the calcium pump. 

PAH, a nonheme iron-containing enzyme, is a member of a larger BI Independent amino acid hydroxylase family. In addition to PAH, the enzyme family includes tyrosine hydroxylase and tryptophan hydroxylase. The enzymes in this family participate in critical metabolic steps and are tissue specific. PAH catabolizes excess dietary PA and synthesizes tyrosine. In adrenal and nervous tissue, tyrosine hydroxylase catalyzes the initial steps in the synthesis of dihydrox-yphenylalanine. In the brain, tryptophan is converted to 5-hydroxytryptophan as the first step of serotonin synthesis. Consequently, these enzymes are highly regulated not only by their expression in different tissues but also by reversible phosphorylation of a critical serine residue found in regulatory domains of the three enzymes. Since all three enzymes are phosphorylated and dephosphorylated by different kinases and phosphatases in response to the need for the different synthetic products, it is not unexpected that the exact regulatory signal for each member of the enzyme family is unique. 

FIGURE 1.1 HTS enzyme assay concepts for kinase and phosphatase screening. 

In contrast to enzyme assays, cell-based assays present the target in a more physiological milieu. With enzyme assays, it may be difficult to purify and express active kinases and phosphatases in their full-length forms and they may require the use of fusion proteins with kinase activity domains. Cell-based technologies, on the other hand, present the opportunity to express the targets with regulatory domains included. Furthermore, cell-based assays usually detect only cell-permeable inhibitors and have the potential to identify more unusual mechanisms, as described earlier. 

Can a phosphorylation-dephosphorylation switch be more sensitive to the level of kinase concentration than n = 1 as given in Equation 5.12 We note that the kinetic scheme in Equation (4.7) is obtained under the assumption of no Michaelis-Menten saturation. Since this assumption may not be realistic, let us move on to study the enzyme kinetics in Figure (5.2) in terms of saturable Michaelis-Menten kinetics. The mechanism by which saturating kinetics of the kinase and phosphatase leads to sensitive switch-like behavior is illustrated in Figure 5.4. The reaction fluxes as a function of / (the ratio [S ]/Sc) for two cases are plotted. The first case (switch off)... 

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