Cofactor, acetylation

An interesting point concerns the reversibility of all of the reaction steps shown in Scheme 12.6. This implies that under certain circumstances, the system might change its behaviour from an ATPase mimic to ATP synthase-type behaviour. This has been achieved through the use of a phosphorylating cofactor, acetyl phosphate (AcP, MeC02P032-). ar,d a divalent metal cation as a promoter (Mg2+ or Ca2+). Under... 

The conversion occurs through a multistep sequence of reactions catalyzed by a complex of enzymes and cofactors called the pyruvate dehydrogenase complex. The process occurs in three stages, each catalyzed by one of the enzymes in the complex, as outlined in Figure 29.11 on page 1152. Acetyl CoA, the ultimate product, then acts as fuel for the final stage of catabolism, the citric acid cycle. All the steps have laboratory analogies. 

The preferred substrates of acetyltransferases are amino-groups of antibiotics, like chloramphenicol, strepto-gramin derivatives, and the various aminoglycosides. The modification is believed to block a functional group involved in the drug-target-interaction. All acetyltransferases use acetyl-coenzyme A as cofactor. 

Acetyl-CoA is also utilized as a cofactor to modify chloramphenicol by O-acetyltranferases (CATs). These enzymes have been found in many different bacterial genera and are usually plasmid encoded in clinical isolates. Furthermore, streptogramin type A antibiotics are acetylatedby Vat enzymes that occur on plasmids in staphylococci and enterococci. 

In principle, numerous reports have detailed the possibility to modify an enzyme to carry out a different type of reaction than that of its attributed function, and the possibility to modify the cofactor of the enzyme has been well explored [8,10]. Recently, the possibility to directly observe reactions, normally not catalyzed by an enzyme when choosing a modified substrate, has been reported under the concept of catalytic promiscuity [9], a phenomenon that is believed to be involved in the appearance of new enzyme functions during the course of evolution [23]. A recent example of catalytic promiscuity of possible interest for novel biotransformations concerns the discovery that mutation of the nucleophilic serine residue in the active site of Candida antarctica lipase B produces a mutant (SerlOSAla) capable of efficiently catalyzing the Michael addition of acetyl acetone to methyl vinyl ketone [24]. The oxyanion hole is believed to be complex and activate the carbonyl group of the electrophile, while the histidine nucleophile takes care of generating the acetyl acetonate anion by deprotonation of the carbon (Figure 3.5). 

Pyruvate is oxidized to acetyl-GoA by a multienzyme complex, pyruvate dehydrogenase, that is dependent on the vitamin cofactor thiamin diphosphate. 

This system is present in many tissues, including hver, kidney, brain, lung, mammary gland, and adipose tissue. Its cofactor requirements include NADPfl, ATP, Mn, biotin, and HC03 (as a source of CO2). Acetyl-CoA is the immediate substrate, and free palmitate is the end product. 

The synthesis of CX6 fatty acid from acetyl-CoA requires 1 acetyl-CoA and 7 malonyl-CoA. The synthesis of each malonyl-CoA requires an ATP (and the cofactor biotin). 

Brain ChAT has a KD for choline of approximately 1 mmol/1 and for acetyl coenzyme A (CoA) of approximately 10pmol/l. The activity of the isolated enzyme, assayed in the presence of optimal concentrations of cofactors and substrates, appears far greater than the rate at which choline is converted to ACh in vivo. This suggests that the activity of ChAT is repressed in vivo. Surprisingly, inhibitors of ChAT do not decrease ACh synthesis when used in vivo this may reflect a failure to achieve a sufficient local concentration of inhibitor, but also suggests that this step is not rate-limiting in the synthesis of ACh [18-20]. 

The PDHC catalyzes the irreversible conversion of pyruvate to acetyl-CoA (Fig. 42-3) and is dependent on thiamine and lipoic acid as cofactors (see Ch. 35). The complex has five enzymes three subserving a catalytic function and two subserving a regulatory role. The catalytic components include PDH, El dihydrolipoyl trans-acetylase, E2 and dihydrolipoyl dehydrogenase, E3. The two regulatory enzymes include PDH-specific kinase and phospho-PDH-specific phosphatase. The multienzyme complex contains nine protein subunits, including... 

Coenzyme A (CoA), 20 249—250. See also Ace to acetyl- Co A in citric acid cycle, 6 633 Coenzyme Q10, 17 673 Coercivity, ofM-type ferrites, 11 70 Coextruded food packaging, 18 44, 45 Coextrusion techniques, for gelatin capsule preparation, 11 549 Cofactors, 10 253 11 4 folic acid, 25 801-802 for enzymes, 3 672-673 protein, 20 828-829 vitamin B12, 25 804 vitamins as, 25 781 Coffea arabica, 7 250 Cojfea Canephora, 7 250 Coffea liberica, 7 250 Coffee, 2 108 6 366 7 250-271 biotechnology, 7 265-267 decaffeinated, 7 263 economic aspects, 7 263-264 estimated maximum oxygen tolerance, 3 381t... 

The major substrates for acetylation are primary aromatic amines, hydroxylamines (both the oxygen and the nitrogen can be acetylated), and hydrazines (11). The cofactor is acetyl Co-A, which is a thioester (Fig. 7.6). 

FIGURE 7.6 Acetylation of a substrate containing an NH2 group utilizing acetyl Co-A as a cofactor. 

ATP and magnesium were required for the activation of acetate. Acetylations were inhibited by mercuric chloride suggesting an SH group was involved in the reaction either on the enzyme or, like lipoic acid, as a cofactor. Experiments from Lipmann s laboratory then demonstrated that a relatively heat-stable coenzyme was needed—a coenzyme for acetylation—coenzyme A (1945). The thiol-dependence appeared to be associated with the coenzyme. There was also a strong correlation between active coenzyme preparations and the presence in them of pantothenic acid—a widely distributed molecule which was a growth factor for some microorganisms and which, by 1942-1943, had been shown to be required for the oxidation of pyruvate. 

Figure 13.2. The preferred flow of reductant from aromatic aldehydes to the acetyl-CoA pathway by the acetogen C. formicoaceticum. THF, tetrahydrofolate brackets, the Ci unit is bound to a cofactor or structurally associated with an enzyme.

Figure 3.4 Structure of two prosthetic groups (a) biotin (b) lipoate. Biotin functions as a carboxyl group carrier, e.g. in acetyl-CoA carboxylase. Lipoate is presented in its oxidised form (-S-S-). It is a cofactor for pyruvate dehydrogenase and oxoglu-tarate dehydrogenase.

The sirtuins (silent information regulator 2-related proteins class III HDACs) form a specific class of histone deacetylases. First, they do not share any sequence or structural homology with the other HDACs. Second, they do not require zinc for activity, but rather use the oxidized form of nicotinamide adenine dinucleotide (NAD ) as cofactor. The reaction catalyzed by these enzymes is the conversion of histones acetylated at specific lysine residues into deacetylated histones, the other products of the reaction being nicotinamide and the metabolite 2 -0-acetyl-adenosine diphosphate ribose (OAADPR) [51, 52]. As HATs and other HDACs, sirtuins not only use acetylated histones as substrates but can also deacetylate other proteins. Intriguingly, some sirtuins do not display any deacetylase activity but act as ADP-ribosyl transferases. 

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