Acetyl-CoA carboxylase phosphatase

Dephosphorylation and activation of the carboxylase have been demonstrated by using the endogenous phosphatase (15, 20) as well as exogenous hen oviduct phosphoprotein phosphatase (65) and phos-phorylase phosphatase (41, 60). Finally, homogeneous acetyl-CoA carboxylase phosphatase from epididymal tissues (61) has been obtained recently. The properties of the isolated enzyme will be discussed briefly... 

Fig. 6. Activation and release of P from purified acetyl-CoA carboxylase by acetyl-CoA carboxylase phosphatase. P-labeled acetyl-CoA carboxylase (2.6 x 10 cpm/mg of protein) was incubated for increasing lengths of time with 0.5 units of acetyl-CoA carboxylase phosphatase. Aliquots were withdrawn to assay the carboxylase (O-O) or...

In an attempt to isolate a specific phosphatase for the carboxylase among multiple forms of the phosphatases in the cell, Krakower and Kim 61) purified acetyl-CoA carboxylase phosphatase by utilizing the multienzyme complex nature of acetyl-CoA carboxylase and its phosphatase. This approach assured the isolation of the specific phosphatase for the carboxylase from various other cytosolic phosphatases with broad substrate specificities 9,11,23,31,32,48,49,53,55,56,58,59, 63, 72, 73, 85, 114). 

Acetyl-CoA carboxylase phosphatase was purified as described in the text. To eliminate interference due to the endogenous carboxylase, the phosphatase was separated from the carboxylase in steps 1-4 by treatment with 5% (w/v) polyethylene glycol. Specific activity and total activity were determined at each stage of the purification using PP]phosphoprotamine as the substrate. The amount of tissue used was 100 g. 

Using a similar approach to the isolation of acetyl-CoA carboxylase phosphatase. Lent and Kim (70) isolated a cyclic AMP-independent protein kinase for acetyl-CoA carboxylase (Table IV). The properties of this cyclic AMP-independent protein kinase are different from those reported by Shiao et al. (105). The enzyme had a Km for acetyl-CoA carboxylase of 85 nAf and requires CoA for activity. The significance of this CoA requirement is further discussed in Section V,C. Phos-phorylase b and HMG-CoA reductase do not serve as substrates for this kinase however, protamine, casein, and histones are phosphorylated. The subunit molecular weight, as measured under denaturing conditions, was 160,000, which is similar to that reported by Shiao et al. (105). However, the CoA-requiring enzyme exists in multimers under nondenaturing conditions, with molecular weights between 700,000 and 2 x 10 . 

Fig. 5 Proposed signal transduction mechanisms that stimulate the pheromone biosynthetic pathway in Helicoverpa zea and Bombyx mori. It is proposed that PBAN binds to a G protein-coupled receptor present in the cell membrane that upon PBAN binding will induce a receptor-activated calcium channel to open causing an influx of extracellular calcium. This calcium binds to calmodulin and in the case of B. mori will directly stimulate a phosphatase that will dephosphorylate and activate a reductase in the biosynthetic pathway. In H. zea the calcium-calmodulin will activate adenylate cyclase to produce cAMP that will then act through kinases and/or phosphatases to stimulate acetyl-CoA carboxylase in the biosynthetic pathway...

Orthophosphate as substrate or product, ACETATE KINASE (PYROPHOSPHATE) ACETYL-CoA CARBOXYLASE ACID PHOSPHATASE ACTOMYOSIN ATPase ACYL PHOSPHATASE ASPARTATE-SEMIALDEHYDE DEHYDROGENASE ATPases... 

Shiao, M.-S. Drong, R.F. Porter, J.W. The purification and properties of a protein kinase and the partial purification of a phosphoprotein phosphatase that inactivate and activate acetyl-CoA carboxylase. Biochem. Biophys. Res. Commun., 98, 80-87 (1981)... 

Regulation of acetyl-CoA carboxylase by phosphorylation and dephosphorylation. Glucagon is known to activate cAMP-dependent protein kinase this kinase phosphorylates both serine 77 and serine 1200 of rat acetyl-CoA carboxylase, which inactivates the enzyme. However, there is also an AMP-dependent kinase that phosphorylates serine 79 and serine 1200 and inactivates the rat acetyl-CoA carboxylase. The relative importance of these two kinases in regulating the carboxylase in vivo is still unclear. Likewise, the phosphorylated enzyme is a substrate for several different protein phosphate phosphatases, and the physiologically relevant phosphatases are not known. Epinephrine may inhibit the carboxylase via a Ca2+-dependent protein kinase. 

Glucagon decreases cholesterol synthesis in isolated hepatocytes [131,132] apparently because it reduces the fraction of hydroxymethylglutaryl-CoA reductase in the active form [131,132], This is due to an increase in reductase kinase activity [133], However, there is no evidence that cAMP-dependent protein kinase phos-phorylates either the reductase, reductase kinase or reductase kinase kinase [134], It has been proposed that the phosphorylation state of these enzymes is indirectly controlled through changes in the activity of protein phosphatase I [132,134], This phosphatase can dephosphorylate and activate the reductase [134,135] and its activity can be controlled by a heat stable inhibitor (inhibitor 1), the activity of which is increased by cAMP-dependent phosphorylation [136,137], Since the phosphorylated forms of acetyl-CoA carboxylase, ATP-citrate lyase, pyruvate kinase, phos-phorylase, phosphorylase kinase and glycogen synthase are also substrates for protein phosphatase I [135], this mechanism could also contribute to their phosphorylation by glucagon. 

Global regulation is carried out by means of reversible phosphorylation. Acetyl CoA carboxylase is switched off by phosphorylation and activated by dephosphorylation (Figure 22.26). Modification of a single serine residue by m AMP-dependent protein kinase (AMPK) converts the carboxylase into an inactive form. The phosphoryl group on the inhibited carboxylase is removed by protein phosphatase 2A. The proportion of carboxylase in the active dephosphorylated form depends on the relative rates of these opposing enzymes. 

How is the enzyme dephosphorylated and activated Insulin stimulates the carboxylase by causing its dephospkorylation. It is not clear which of the phosphatases activates the carboxylase in response to insulin. The hormonal control of acetyl CoA carboxylase is reminiscent of that of glycogen synthase (Section 21.5.2). 

Fatty acid synthesis and degradation are reciprocally regulated so that both are not simultaneously active. Acetyl CoA carboxylase, the essential control site, is phosphorylated and inactivated by AMP-dependent kinase. The phosphorylation is reversed by a protein phosphatase. Citrate, which signals an abundance of building blocks and energy, partly reverses the inhibition by phosphorylation. Carboxylase activity is stimulated by insulin and inhibited by glucagon and epinephrine. In times of plenty, fatty acyl CoAs do not enter the mitochondrial matrix, because malonyl CoA inhibits carnitine acyl transferase I. 

Fig. 33.11. Regulation of acetyl Co A carboxylase. This enzyme is regulated allosterically, both positively and negatively, by phosphorylation (circled P) and dephosphorylation, and by diet-induced induction (circled t). It is active in the dephosphorylated state when citrate causes it to polymerize. Dephosphorylation is catalyzed by an insulin-stimulated phosphatase. Low energy levels, via activation of an AMP-dependent protein kinase, cause the enzyme to be phosphorylated and inactivated. The ultimate product of fatty acid synthesis, palmitate, is converted to its CoA derivative palmityl CoA, which inhibits the enzyme. A high-calorie diet increases the rate of transcription of the gene for acetyl CoA carboxylase, whereas a low-calorie diet reduces transcription of this gene.

Fig. 36.5. Regulation of acetyl CoA carboxylase (AcC). AcC is regulated by activation and inhibition, by phosphorylation (mediated by the AMP-activated protein kinase) and dephosphorylation (via an insulin-stimulated phosphatase), and by induction and repression. It is active in the fed state.

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). 

Reports that the activation of acetyl-CoA carboxylase from rat epididymal fat tissues 60) and rabbit mammary glands 41) by liver phosphorylase phosphatase is accompanied by dephosphorylation, and that both events are blocked by rat liver phosphatase inhibitor-1, provide compelling evidence for the occurrence of the covalent modification mechanism in the control of the activity of the carboxylase. However, isolation and studies of the properties of specific enzymes for the interconversion are essential for our understanding of the mechanism... 

Figure 2.11. Lipid synthesis. GDP = glycerol-3-phosphate dehydrogenase. PP = phosphat-idate phosphatase. CPT=choline phosphotransferase. BPT= ethanolamine phosphotransferase. AC = acetyl CoA carboxylase. HAD 3-hydroxyacyl-CoA dehydrogenase. CCPT = cemmide choline phosphotransferase. HMGR = hydroxymethyl utaryl-CoA reductase. MK = mevalonate kinase. IPPI = isopentenyl pyrophosphate isomerase...

The insulin receptor itself is a protein kinase, which phosphorylates susceptible tyrosine residues in proteins. When insulin binds to the external part of the receptor, there is a conformational change in the whole of the protein, resulting in activation of the protein kinase region at the inner face of the membrane. This phosphorylates, and activates, cytosolic protein kinases, which, in turn, phosphorylate target enzymes, including phosphoprotein phosphatase (see Figure 10.6), cAMP phosphodiesterase (see Figure 10.8) and acetyl CoA carboxylase. 

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