Identity of Malonyl and Palmitoyl Transferase of Fatty Acid Synthetase from Yeast. 1. Functional Interrelationships between the Acyl Transferases

Abstract

Functional interrelationships between the acyl transferases of yeast fatty acid synthetase were investigated. In binding assays with synthetase modified by 5,5'-dithiobis(2-nitrobenzoic acid), 4 -5 malonyl transferase entities per multienzyme complex molecule could be titrated. In the presence of palmitoyl-CoA these malonyl transferases were found inaccessible to malonyl-CoA, whereas the acetyl transferases were reactive towards acetyl-CoA.Between four and five palmitoyl transferase entities per synthetase equivalent were found reactive towards palmitoyl-CoA, the palmitoyl binding being inhibited by malonyl-CoA. Following palmitoyl binding the acetyl transferases were found reactive towards acetyl-CoA.Substrate model assays were consistent with these data. It is concluded that malonyl and palmitoyl transferases are closely coupled enzyme components of the multienzyme complex which are fairly independent of the acetyl transferase entities. The molecular basis for the observed coupling will be given in the following paper.Fatty acid synthetase from yeast is a stable multienzyme complex ( M , = 2.3 x lo6) which catalyzes the formation of long-chain fatty acyl-coenzyme A compounds according to Eqn (1) :( 1) where n = 7.8. As shown in this laboratory [l], an acetyl residue derived from acetyl-CoA in a recurrent reaction sequence is elongated by successive addition of C2 carbon units derived from malonyl-CoA. Intermediates of the biosynthetic process are never released * Deceased.

Substrate and Product Binding Sites of Yeast Fatty Acid Synthase. Stoichiometry and Binding Kinetics of Wild-Type and in vitro Mutated Enzymes

“…In wild-type FAS, only one out of six SHp sites and two of the six OHMal sites were occupied with malonate. These values are in good agreement with earlier though less systematically performed experiments from Lynen's laboratory [5, 6,9,211. Thus, there can be no doubt about the lack of full-site reactivity of yeast fatty acid synthase with its substrates, acetyl-CoA and malonyl-CoA.…”

Animal Fatty Acid Synthase: A Chemical Nanofactory

“…In 1970, Phillips and co-workers reported that a hydroxyl site of the animal (pigeon) FAS transfers the CoA acetyl and malonyl moieties to a sulfhydryl acceptor (i.e., the PNS group of the ACP) . Later that year, Joshi et al tentatively identified the hydroxyl site as the hydroxyl group of a serine residue, similarly to what was reported for the bacterial ( E. coli ) malonyltransferase as well as for the fungal (yeast) malonyltransferase domain. , Moreover, the observation that acetyl and malonyl groups bound to the hydroxyl site of the animal (pigeon) FAS and formed stable intermediates indicates that the acyl moieties are, in a first stage, transferred to the catalytic serine of the MAT domain of FAS and, in a second stage, loaded into the ACP domain. , This serine-centered, two-stage transfer reaction is consistent with a ping-pong bi-bi mechanism that is shared among the AT enzymes/domains of all FAS types. ,,,,− …”

“…The fungal/bacterial FAS exhibit two different AT domains/enzymes. One of them transfers the primer acyl substrate, and the other transfers the extender acyl substrates to the ACP. − The AT domain of animal FAS, MAT, is more versatile, being capable of loading both the acetyl starter and malonyl extender molecules. ,,, Past studies have reported that the acetyl-CoA and malonyl-CoA substrates randomly bind to a common active site of the FAS (i.e., the MAT domain) which, consequently, makes them competitive inhibitors of each other’s binding. ,,− Thus, for an efficient fatty acid synthesis, the inappropriately bound acetyl or malonyl moiety must be rapidly unloaded (i.e., the acyl-FAS bond must be hydrolyzed) and transferred back to the CoA until a competent enzyme complex is formed. , The dual specificity of MAT makes it responsible for initiating the fatty acid synthesis reaction and for continuously providing the C 2 extender substrate (i.e., malonyl moiety) at each elongation cycle. It is noteworthy that, in rare circumstances or in certain specialized tissues, the MAT domain is capable of accepting unusual CoA-esters substrates such as propionyl-, methylmalonyl-, and phenylacetyl-CoA, and of incorporating them in the fatty acid synthesis.…”

Identification of genes required for Pseudomonas aeruginosa carnitine catabolism

“…We measured carnitine-dependent CoA utilization in cytoplasmic extracts using the 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) method (Engeser et al, 1979). Reaction conditions were: RB plus 4 mM NAD + , 4 mM ATP, 1 mM CoA, 25 mg cytoplasmic extract ml 21 , and with 50 mM L-carnitine or an equivalent volume of 0.5 M HEPES, pH 7.3.…”