Acetyl phosphatase and

Figure 2. Mechanism of PDH. The three different subunits of the PDH complex in the mitochondrial matrix (E, pyruvate decarboxylase E2, dihydrolipoamide acyltrans-ferase Ej, dihydrolipoamide dehydrogenase) catalyze the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2. E, decarboxylates pyruvate and transfers the acetyl-group to lipoamide. Lipoamide is linked to the group of a lysine residue to E2 to form a flexible chain which rotates between the active sites of E, E2, and E3. E2 then transfers the acetyl-group from lipoamide to CoASH leaving the lipoamide in the reduced form. This in turn is oxidized by E3, which is an NAD-dependent (low potential) flavoprotein, completing the catalytic cycle. PDH activity is controlled in two ways by product inhibition by NADH and acetyl-CoA formed from pyruvate (or by P-oxidation), and by inactivation by phosphorylation of Ej by a specific ATP-de-pendent protein kinase associated with the complex, or activation by dephosphorylation by a specific phosphoprotein phosphatase. The phosphatase is activated by increases in the concentration of Ca in the matrix. The combination of insulin with its cell surface receptor activates PDH by activating the phosphatase by an unknown mechanism.

There are many examples of phosphorylation/dephosphorylation control of enzymes found in carbohydrate, fat and amino acid metabolism and most are ultimately under the control of a hormone induced second messenger usually, cytosolic cyclic AMP (cAMP). PDH is one of the relatively few mitochondrial enzymes to show covalent modification control, but PDH kinase and PDH phosphatase are controlled primarily by allosteric effects of NADH, acetyl-CoA and calcium ions rather than cAMP (see Table 6.6). 

Minhas T and Greenman J (1989) Production of cell-bound and vesicle-associated trypsin-like protease, alkaline phosphatase and N-acetyl—glucosaminidase by Bacteroides gingivalis strain W50. J Gen Microbiol 135, 557-564. 

Interconversion processes (see p. 120) also play an important role. They are shown here in detail using the example of the PDH complex (see p. 134). The inactivating protein kinase [la] is inhibited by the substrate pyruvate and is activated by the products acetyl-CoA and NADH+H. The protein phosphatase [Ibj—like isodtrate dehydrogenase [3] and the ODH complex [4j-is activated by Ca. This is particularly important during muscle contraction, when large amounts of ATP are needed. Insulin also activates the PDH complex (through inhibition of phosphorylation) and thereby promotes the breakdown of glucose and its conversion into fatty acids. 

In subsequent years, much evidence has been adduced to support this mechanism. Alkaline phosphatase and, by analogy, other serine enzymes, are directly phosphorylated on serine serine phosphate is not an artifact (Kennedy and Koshland, 1957). In the presence of nitrophenyl acetate, chymotrypsin is acetylated on serine, and the resulting acetylchymotrypsin has been isolated (Balls and Aldrich, 1955 Balls and Wood, 1956). Similarly, the action of p-nitrophenyl pivalate gave rise to pivaloyl chymotrypsin, which could be crystallized (Balls et al., 1957). Neurath and workers showed that acetylchymotrypsin is hydrolyzed at pH 5.5, but that it is reversibly denatured by 8 M urea the denatured derivative is inert to hydrolysis and even to hydroxylamine, whereas the renatured protein, obtained by... 

Regulation of the Pyruvate Dehydrogenase Complex In animal tissues, the rate of conversion of pyruvate to acetyl-CoA is regulated by the ratio of active, phosphory-lated to inactive, unphosphorylated PDH complex. Determine what happens to the rate of this reaction when a preparation of rabbit muscle mitochondria containing the PDH complex is treated with (a) pyruvate dehydrogenase kinase, ATP, and NADH (b) pyruvate dehydrogenase phosphatase and Ca2+ (c) malonate. 

In maize-root tips, high specific activities of/ -D-galactosidase, a- and jff-D-glucosidase, N-acetyl-/ -D-glucosaminidase, acid phosphatase, and phosphodiesterase (EC 3.1.4.1) are found in the cell-wall fraction.246... 

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. 

Animal and bacterial enzymes that utilize or synthesize carbamyl phosphate have activity with acetyl phosphate. Acyl phosphatase hydrolyzes both substrates, and maybe involved in the specific dynamic action of proteins. Ornithine and aspartic transcarbamylases also synthesize acetylornithine and acetyl aspartate. Finally, bacterial carbamate kinase and animal carbamyl phosphate synthetase utilize acetyl phosphate as well as carbamyl phosphate in the synthesis of adenosine triphosphate. The synthesis of acetyl phosphate and of formyl phosphate by carbamyl phosphate synthetases is described. The mechanism of carbon dioxide activation by animal carbamyl phosphate synthetase is reviewed on the basis of the findings concerning acetate and formate activation. 

While quite apparent to us now, it has taken many years to realize that acetyl-P and carbamyl-P could be substrates for the same enzymes. Acetyl-P was considered, for some years, an important intermediate for acetylation. However, no evidence for either its synthesis or its utilization, other than by phosphatase action (26) with animal... 

Heat stability and other characteristics indicate that this enzyme may be identical with the acetyl phosphatase of Lippman (26), which also catalyzes the hydrolysis of 1,3-diphosphoglycerate (21). 

In LLC-PKj cells gentamicin induces membrane damage as shown by the loss of specific membrane enzymes (y-glutamyl transpeptidase, alkaline phosphatase and aminopeptidase), a decrease of the lysosomal enzyme N-acetyl-P-D-glucosaminidase, an inhibition of apical Na -dependent glucose transporter and the basolateral Na-K-ATPase pump as well as a decrease in dome formation [141, 142]. Furthermore gentamicin results in a dose dependent decrease in intracellular ATP and cAMP [142]. 

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