Malonyl binding

Fig. 8. A. Free energy profile for Na+ movement through the malonyl Gramicidin channel. Calculated using Eyring rate theory and the locations of binding sites in the channel.

Fig. 10. Calculated sodium ion single channel currents for the malonyl Gramicidin channel and comparison with experimental data points using four different models all of which fit the data well but only one of which, B., is correct. The point to be made is that both the independent determination of rate constants and of the binding site locations are required.

Following the formation of malonyl CoA, another nucleophilic acyl substitution reaction occurs in step 4 to form the more reactive malonyl ACP, thereby binding the malonyl group to an ACP arm of the multienzyme synthase. At this point, both acetyl and malonyl groups are bound to the enzyme, and the stage is set for their condensation. 

Murthy, M.S.R. Pande, S.V. (1987). Malonyl-CoA binding site and the overt carnitine palmitoyltransferase activity reside on the opposite sides of the outer mitochondrial membrane. Proc. Nat. Acad. Sci. USA 84,378-382. 

Thiolactomycin (16) is another natural product that reversibly inhibits E. coli FabF, FabB, and FabH with respective ICso s of 6, 25 and 110 (iM. Unlike cerulenin, it binds the malonyl-ACP site of the enzyme [27]. Despite modest double-digit MICs on . coli, S. aureus, Serratia marces-cens, and Mycobacterium tuberculosis, 16 has generated quite some interest due to its good in vivo protection against an oral or intramuscular S. marcescens urinary tract infection model where it displayed rapid tissue distribution [28]. Despite several medicinal chemistry efforts, thiolactomycin has proven difficult to optimize due to some strict functional group requirements for its SAR [29]. 

The way biotin participates in carbon dioxide fixation was established in the early 1960s. In 1961 Kaziro and Ochoa using propionyl CoA carboxylase provided evidence for 14C02 binding in an enzyme-biotin complex. With excess propionyl CoA the 14C label moved into a stable position in methyl malonyl CoA. In the same year Lynen found biotin itself could act as a C02 acceptor in a fixation reaction catalyzed by B-methylcrotonyl CoA carboxylase. The labile C02 adduct was stabilized by esterification with diazomethane and the dimethyl ester shown to be identical with the chemically synthesized molecule. X-ray analysis of the bis-p-bromanilide confirmed the carbon dioxide had been incorporated into the N opposite to the point of attachment of the side chain. Proteolytic digestion and the isolation of biocytin established the biotin was bound to the e-NH2 of lysine. 

The fatty acid synthase protein is known to contain an acyl carrier protein (ACP) binding site, and also an active-site cysteine residue in the P-ketoacyl synthase domain. Acetyl and malonyl gronps are successively transferred from coenzyme A esters and attached to the thiol groups of Cys and ACP. 

Malonate binds to the ph osphopanthotheine coenzyme site and then the acetyl or acyl group is transferred to carbon two of malonate, with the loss of one malonyl carbon as C02-... 

To study the entry of 6-hydroxymelein synthase to the substrate, three functional groups at the reaction center of the enzyme, Ser-OH, Cys-SH and ACP-SH, were alkylated appropriately, and the binding abilities of the modified proteins toward the substrates were examined [83]. It appeared that cysteine-SH accepted only the acetyl group, while cysteamine-SH of ACP was preferentially malonylated in the presence of both substrates. A plausible explanation for the initiation of the synthase... 

After malonyl-CoA synthesis, the remaining steps in fatty acid synthesis occur on fatty acid synthase, which exists as a multienzyme complex. In the initial reactions acetyl-CoA and malonyl-CoA are transferred onto the protein complex by acetyl-CoA transacylase and malonyl-CoA transacylase (step 1 and step 2 in fig. 18.12a). The acceptor for the acetyl and malonyl groups is acyl carrier protein (ACP). ACP also carries all of the intermediates during fatty acid biosynthesis. The prosthetic group that binds these intermediates is... 

CoA to form malonyl CoA using C02 in the form of bicarbonate HC03 (Fig. 2). This reaction is catalyzed by the enzyme acetyl CoA carboxylase which has biotin as a prosthetic group, a common feature in C02-binding enzymes. One molecule of ATP is hydrolyzed in the reaction, which is irreversible. The elongation steps of fatty acid synthesis all involve intermediates linked to the terminal sulfhydryl group of the phosphopantetheine reactive unit in ACP phosphopantetheine is also the reactive unit in CoA. Therefore, the next steps are the formation of acetyl-ACP and malonyl-ACP by the enzymes acetyl transacylase and malonyl transacylase, respectively (Fig. 2). (For the synthesis of fatty acids with an odd number of carbon atoms the three-carbon propionyl-ACP is the starting point instead of malonyl-ACP.)... 

The formation of malonyl-CoA signals the beginning of the synthesis of palmitic acid (C16 ()). This occurs on a multifunctional enzyme complex, the fatty acid synthase. In mammalian liver, the enzyme complex consists of two identical polypeptides, each with specific binding sites for malonyl and alkanoyl groups, and eight different enzyme activities. 

The malonyl and alkanoyl binding sites on the fatty acid synthase are as follows ... 

The 2-pyrone synthase (2-PS) found in several plant species is the simplest Type III PKS.ii i 2-PS condenses three malonyl-CoAs and yields a spontaneously cyclized triketide lactone (TKL). The first malonyl-CoA is decarboxylated and serves as the acetate primer, analogous to the decarboxylative mechanism observed in Type II PKSs. The 2-PS is unable to accept larger starter units due to a considerable smaller active site than that of CHS. Mutating the residues that surround the CHS-binding pocket to those found in 2-PS yielded a mutant CHS that is biochemically indistinguishable from 2-PS." ... 

Mancia, F., and Evans, P. R., 1998, Conformational changes on substrate binding to methyl-malonyl CoA mutase and new insights into the free radical mechanism. Structure 6 7119720. 

---