Regulation of Enzyme Activity by Phosphorylation

Binding of the inhibitor to the enzyme may alter the orientation of the catalytically essential groups in a way that does not allow efficient catalysis and/or strong binding of the substrate. Examples are the inhibitors of the cyclin-dependent protein kinases of the cell cycle (see Chapter 13). 

Inhibitor proteins themselves are subject to a variety of regulation mechanisms. The function of an inhibitor protein can be regulated, for example, by protein phosphorylation (see Section7.6.2 ), by degradation or by de novo synthesis (see Chapter 13). 

Examples of the reversible association of activator proteins with an enzyme are the Ca2+-calmodulin- dependent enzymes. Calmodulin is a Ca2+-binding protein which can activate target enzymes, e.g., phosphorylase kinase (see Section 6.7.1 and Section 7.5) in its Ca2+-bound form. Other examples of activating proteins are the cyclins (see Chapter 13). The cyclins are activators of protein kinases that regulate the cell cycle. Structural studies have shown that the cyclins activate the protein kinases of the cell cycle by restructuring the active site and positioning the catalytic groups to allow efficient catalysis. 

Activator proteins themselves can be bound in regulatory networks, as shown in the example of the cyclins (Chapter 13). The function of an activator protein can be regulated at the level of gene expression, degradation, or post-translational modification (e.g., protein phosphorylation). 

The availability of metal ions can also be employed for regulation of enzyme activity. Of primary importance is Ca2+. An important example in this regard is protein kinase C, which is activated by Ca2+ (see Section 7.4). The availability of Ca2+ is further regulated in various ways by hormone-controlled pathways (see Chapter 6). 

The phosphorylation of enzymes by specific protein kinases is a widespread mechanism for the regulation of enzyme activity. It represents a flexible and reversible means of regulation and plays a central role in signal transduction chains in eucaryotes. 

Proteins are phosphorylated mainly on Ser/Thr residues and on Tyr residues. Occasionally Asp or His residues are phosphorylated, the latter especially in procaryotic signal transduction pathways (see chapter 7, chapter 12). For the regulation of enzyme activity the phosphorylation of Ser and Thr residues is most significant. Apart from regulation of Tyr kinases, Tyr phosphorylation serves the function of creating specific attachment sites for proteins. Both of these functions will be discussed in more detail in chapter 8. 

Protein phosphorylation is a specific enzymatic reaction in which one protein serves as a substrate for a protein kinase. Protein kinases are phophotransferases. They catalyze the transfer of a phosphate group from ATP to an acceptor amino acid in the substrate protein (fig. 2.10). A detailed discussion of protein kinases can be found in chapter 7. 

The phosphate ester of Ser, Thr, or Tyr residues are quite stable at room temperature and neutral pH the rate of spontaneous hydrolysis is very low. Therefore, to remove the phosphate residue the cell utilizes specific enzymes termed phosphatases. Based on substrate specificity, these can be classified as Ser-, Thr- or Tyr-specific phosphatases (see chapter 8). 

---

The examples of phosphorylase kinase and protein phosphatase I illustrate some important principles of regulation of enzyme activity by phosphorylation and dephosphorylation events. They clearly indicate how different signal transduction paths can meet in key reactions of metabolism, how signals can be coordinated with one another and how common components of a regulation network can be activated by different signals. The following principles are highlighted ... 

Direct regulation of enzyme activities by ATP, ADP, NADH, and other effectors Indirect regulation through obligatory coupling of oxidation wtih phosphorylation ... 

In addition, PolyPs are most likely involved in the regulation of enzyme activities by participation in their phosphorylation. A protein phosphorylation process, using not ATP but high-polymer PolyPs, was revealed in the archae Sulfolobus acidocaldarius (Skorko, 1989). Tripolyphosphate was observed to be a phosphodonor of selective protein phosphorylation of rat liver microsomal membrane (Tsutsui, 1986). 

The regulation of eNOS activity by phosphorylation is a well documented subject as the eNOS protein possess several consensus sequences for phosphorylation by protein kinase A (PKA), protein kinase C (PKC), protein kinase B (Akt/PKB) and AMP-activated protein kinase (AMPK). The eNOS enzyme has been reported to be phosphorylated on threonine, serine and t50 osine residues in response to various agonists. Many studies have shown that phosphorylation of eNOS on serine 1177 leads to an activation of eNOS [26-29], whereas phosphorylation on threonine 495 inactivates eNOS as this site is in the Ca Vcalmodulin binding domain [26]. Fleming et al. (2001) demonstrated that bradykinin (100 nM) increased eNOS activity in both porcine coronary artery endothelial cells (PCAE) and HUVEC via dephosphorylation of threonine 495 and phosphorylation of serine 1177 [30]. The bradykinin-induced phosphorylation of serine 1177 was abolished in the presence of a calmodulin dependent kinase II inhibitor whilst the dephosphorylation of threonine 495 was abolished by a protein phosphatase I inhibitor [30]. Harris et al. (2001) documented in BAEC that bradykinin (1 pM)-induced eNOS activity was mediated by activation of Akt/PKB [31] (see section 1.2.2) resulting in NOS phosphorylation at serine 1179 (bovine sequence) and a de-phosphorylation at threonine 497 mediated by calcineurin phosphatase. Typically, phosphorylation of eNOS at either of these sites is coordinated with dephosphorylation at the alternate site. 

Beg, Z.H. Stonik, J.A. Brewer, B. Human hepatic 3-hydroxy-3-methylglu-taryl coenzyme A reductase evidence for the regulation of enzymic activity by a bicyclic phosphorylation cascade. Biochem. Biophys. Res. Commun., 119, 488-498 (1984)... 

Regulation of enzyme activity by reversible protein phosphorylation... 

Several key questions remain with regard to the regulation of tyrosine hydroxylase by phosphorylation. What is the precise effect of the phosphorylation of each of these serine residues on the catalytic activity of the enzyme How does the phosphorylation of multiple residues affect enzyme activity Does the phosphorylation of one residue affect the ability of the others to be phosphorylated Tyrosine hydroxylase provides a striking example as to how multiple intracellular messengers and protein kinases converge functionally through the phosphorylation of a single substrate protein. Phosphorylation of tyrosine hydroxylase by cAMP-dependent and Ca2+-dependent protein kinases and by MAPK cascades... 

The molecular basis for regulation of enzymatic activity through phosphorylation and dephosphorylation has been established in many enzyme systems (29). The significance of these reactions in histones, ribosomal proteins and KNA polymerase is not known. In an attempt to establish the specificity of the cyclic AMP-dependent protein kinases, the structure of several substrates have been determined (30). The data indicate that the sequence around the phosphorylated serine residue all contain two basic amino acids separated by no more than two residues from the N-terminal of the susceptible serine (e.g. -Arg-Arg-X-Y-Ser-). 

Covalent modification is a major mechanism for the rapid and transient regulation of enzyme activity. Numerous enzymes of intermediary metabolism are affected by phosphorylation, either positively or negatively. Covalent phosphorylations can be reversed by a separate subclass of enzymes known as phosphatases. The aberrant phosphorylation of growth factor and hormone receptors, as well as of proteins that regulate cell division, often leads to unregulated cell growth or cancer. The usual sites for phosphate addition to proteins are the serine, threonine and tyrosine R-group hydroxyl residues. 

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. 

As might be expected from other mechanisms of regulation described in this text, phosphorylation and dephosphorylation of key proteins is the main mechanism for regulating the cycle, i.e. reversible phosphorylation, also known as interconversion cycles (discussed in Chapter 3). In the cell cycle, several of these interconversion cycles play a role in control at the checkpoints. Two important terms must be appreciated to help understand the mechanism of regulation of the cycle the phosphorylation of proteins is catalysed by specific protein kinases, known as cell-division kinases (cdck) or cell cycle kinases (cck) and these enzymes are activated by specific proteins, known as cyclins. 

Figure 20.31 The principle of interconversion cycles in regulation of protein activity or changes in protein concentration as exemplified by translation/proteolysis or protein kinase/protein phosphatase. They result in very marked relative changes in regulator concentration or enzyme activity. The significance of the relative changes (or sensitivity in regulation) is discussed in Chapter 3. The principle of regulation by covalent modihcation is also described in Chapter 3. The modifications in cyclin concentration are achieved via translation and proteolysis, which, in effect, is an interconversion cycle. For the enzyme, they are achieved via phosphorylation and dephosphorylation reactions. In both cases, the relative change in concentration/activity by the covalent modification is enormous. This ensures, for example, that a sufficient increase in cyclin can occur so that an inactive cell cycle kinase can be converted to an active cell cycle kinase, or that a cell cycle kinase can be completely inactivated. Appreciation of the common principles in biochemistry helps in the understanding of what otherwise can appear to be complex phenomena.

The progression of the cell cycle is regulated by interconversion processes, in each phase, special Ser/Thr-specific protein kinases are formed, which are known as cyclin-depen-dent kinases (CDKs). This term is used because they have to bind an activator protein (cyclin) in order to become active. At each control point in the cycle, specific CDKs associate with equally phase-specific cyclins. if there are no problems (e.g., DNA damage), the CDK-cyclin complex is activated by phosphorylation and/or dephosphorylation. The activated complex in turn phosphorylates transcription factors, which finally lead to the formation of the proteins that are required in the cell cycle phase concerned (enzymes, cytoskeleton components, other CDKs, and cyclins). The activity of the CDK-cyclin complex is then terminated again by proteolytic cyclin degradation. 

Fig. 7.17. Regulation of protein phosphatases by inhibitor proteins. The substrates of protein kinase A include protein phosphatase inhibitors that are phosphorylated by the C subunit of protein kinase A. In the phosphorylated state, the protein phosphatase inhibitors bind to the protein phosphatase and inhibit its enzyme activity...

---