Acetyl-CoA binding sites

The protomer consists of four polypeptide chains one of which contains the covalently linked biotinyl prosthetic group [180,206], Each protomer has one tight citrate-binding site (Ad = 3 fiM) and one tight acetyl-CoA binding site (Ad = 4 fiM) [180]. 

Citrate synthase in mammals is a dimer of 49-kD subunits (Table 20.1). On each subunit, oxaloacetate and acetyl-CoA bind to the active site, which lies in a cleft between two domains and is surrounded mainly by a-helical segments (Figure 20.6). Binding of oxaloacetate induces a conformational change that facilitates the binding of acetyl-CoA and closes the active site, so that the reactive carbanion of acetyl-CoA is protected from protonation by water. 

CODH from acetogens found to catalyze acetyl-CoA synthesis and nickel proposed to be the active site of CO oxidation and acetyl-CoA synthesis. CODH found to contain a heterometallic cluster consisting of nickel and iron that binds CO and proposed to be the active site of acetyl-CoA synthesis. Growth of acetogens on nitrate disables the acetyl-CoA pathway. ... 

Figure 6.1 Synthesis and metabolism of acetylcholine. Choline is acetylated by reacting with acetyl-CoA in the presence of choline acetyltransferase to form acetylcholine (1). The acetylcholine binds to the anionic site of cholinesterase and reacts with the hydroxy group of serine on the esteratic site of the enzyme (2). The cholinesterase thus becomes acetylated and choline splits off to be taken back into the nerve terminal for further ACh synthesis (3). The acetylated enzyme is then rapidly hydrolised back to its active state with the formation of acetic acid (4)...

That this is not the case for the enzyme citrate synthase suggests we must look at the enzyme binding site to rationalize the different reaction sequence. It becomes clear that the enzyme binding site positions the substrates so that there are acidic and basic amino acid residues available to produce the enolate anion equivalent of acetyl-CoA (shown here as the enol), but not for the oxaloacetate (Figure 13.8). 

The enzyme is a hexamer, actually a dimer of trimers made up of 291-residue polypeptide chains.28 Aceto-acetyl-CoA is a competitive inhibitor which binds into the active site and locates it. From the X-ray structure of the enzyme-inhibitor complex it can be deduced that the carboxylate group of E144 abstracts a proton from a water molecule to provide the hydroxyl ion that binds to the P position (Eq. 13-6, step a) and that the E164 carboxyl group donates a proton to the intermediate enolate anion in step b.28 The hydroxyl group... 

As seen in the graph, succinyl-CoA shifts the half-saturation point, [S]oe (or K0 ), for acetyl-CoA to the right but does not alter Vmax. This indicates that succinyl-CoA acts as a negative modulator, either directly as a competitive inhibitor with acetyl-CoA or by binding to a site separate from the active site. 

There are many other questions that need to be addressed. For example What are the kinetics of the inhibition Do the different inhibitors bind at the same site What are the molecular requirements for inhibition What are the differences between susceptible and tolerant ACCases and so on. ACCase purified 40 to 100 fold may not be sufficiently pure to answer many of these questions. For example, an extract purified on a Sephacryl S-300 column can have a specific activity up to 400 nmol/min/mg. We have observed that this preparation can catalyze the carboxylation of other short chained acyl CoA s in addition to acetyl CoA (Table VI). Both haloxyfop and tralkoxydim inhibit the carboxylation reaction regardless of whether n-propionyl CoA or acetyl CoA are substrates either individually or together (Table VII). At present, we are unsure whether n-propionyl CoA can be used as a substrate for ACCase or whether a n-propionyl CoA carboxylase is present in the preparation and the herbicides also inhibit that enzyme. 

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