Covalent inhibition, protein kinase

Pyruvate kinase possesses allosteric sites for numerous effectors. It is activated by AMP and fructose-1,6-bisphosphate and inhibited by ATP, acetyl-CoA, and alanine. (Note that alanine is the a-amino acid counterpart of the a-keto acid, pyruvate.) Furthermore, liver pyruvate kinase is regulated by covalent modification. Flormones such as glucagon activate a cAMP-dependent protein kinase, which transfers a phosphoryl group from ATP to the enzyme. The phos-phorylated form of pyruvate kinase is more strongly inhibited by ATP and alanine and has a higher for PEP, so that, in the presence of physiological levels of PEP, the enzyme is inactive. Then PEP is used as a substrate for glucose synthesis in the pathway (to be described in Chapter 23), instead... 

Figure 4.4 Covalent inhibition of protein kinases. Upper part, scheme for a Michael addition reaction. Lower part, structure of a reactive inhibitor, WZ4002 (4.9).

Phosphofructokinase (PFK) purified from A. suum muscle is inhibited by ATP and stimulated by AMP, K", NH2, phosphate, and hexose bisphosphate (19). It has been estimated that the A. suum PFK functions at 1-2% of its maximal velocity in vivo. Both phosphorylated and dephosphorylated forms of the A. suum PFK exhibit similar specific activities, but the dephosphorylated enzyme has a 2-2.5-fold lower activity in a physiological assay system. Phosphorylation of the dephosphorylated PFK with the mammalian cAMP-dependent protein kinase results in the incorporation of about 1 mol of phosphate/mol of subunit and completely restores activity in the physiological assay system (20). Therefore, it appears that PFK activity in A. suum muscle can be regulated, not only by metabolites, but also by covalent modification catalyzed by a cAMP-dependent protein kinase. This contrasts with mammalian muscle, where the effect of PFK phosphorylation is poorly defined and ranges from inhibition to mild stimulation (20). 

ACC-2 produces malonyl CoA, which inhibits carnitine palmitoyl transferase I, thereby blocking fatty acid entry into the mitochondria. Muscle also contains malonyl CoA decarboxylase, which catalyzes the conversion of malonyl CoA to acetyl CoA and carbon dioxide. Thus, both the synthesis and degradation of malonyl CoA is carefully regulated in muscle cells to balance glucose and fatty acid oxidation. Both allosteric and covalent means of regulation are employed. Citrate activates ACC-2, and phosphorylation of ACC-2 by the adenosine monophosphate (AMP)-activated protein kinase inhibits ACC-2 activity. Phosphorylation of malonyl CoA decarboxylase by the AMP-activated protein kinase activates the enzyme, further enhancing fatty acid oxidation when energy levels are low. 

Matsuo M, Reardon S, Ikebe M, Kitazawa T (1994) A novel mechanism for the Ca -sensitizing effect of protein kinase C on vascular smooth muscle inhibition of myosin light chain phosphatase. J Gen Physiol 104 265-286 McDaniel NL, Rembold CM, Murphy RA (1994) Cyclic nucleotide dependent relaxation in vascular smooth muscle. Can J Physiol Pharmacol 72 1380-1385 Murphy RA (1994) What is special about smooth muscle The significance of covalent crossbridge regulation. FASEB J 8 311-318... 

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