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Interaction of pyridoxal phosphate with acetyl-CoA carboxylase Elliott, Jason Edward

Abstract

Acetyl-CoA carboxylase (ACC) catalyzes the conversion of acetyl-CoA to malonyl- CoA, the first committed step in de novo fatty acid synthesis. Two mammalian ACC isoforms exist, with many distinctive properties and differential tissue distribution. ACC isoforms exist as inactive dimers that can be allosterically activated by citrate; activation being associated with polymerization, in the case of ACC-1. Inhibition of other citrateutilizing enzymes by pyridoxal phosphate (PLP) suggested that PLP might also inhibit ACC. This thesis describes work done to assess the interaction of PLP with ACC. PLP was found to be a potent inhibitor of ACC, with an apparent IC50 of 225 u M against ACC purified from liver. The effects of PLP were seen in affinity-purified ACC, suggesting its effects are direct. PLP is effective against both ACC isoforms, but ACC-1 shows greater sensitivity to PLP than ACC-2. PLP inhibition of ACC is rapid, occurring within 1 minute of addition. Pre-treatment of ACC with citrate provided substantial protection against PLP inhibition at low inhibitor concentrations; the protection being more evident with ACC-1 than ACC-2. This suggests PLP and citrate bind to the same site on ACC. Treatment with PLP and sodium borohydride leads to irreversible ACC inhibition, confirming that PLP forms a Schiff base with ACC that can be reduced with sodium borohydride. In the absence of borohydride reduction PLP interacts reversibly with ACC. Selective labelling of ACC using [3H]-borohydride indicated that PLP reacts directly with the ACC subunits. Numerous analogs of PLP were tested for inhibitory action against ACC. From these studies it was concluded that the aldehyde and negative charges on PLP are especially important for its inhibitory action on ACC. The pyridine ring is also important, but to a lesser degree than the aldehyde or phosphate. The ability to use PLP to determine the sequence of the citrate binding site and to provide the basis for inhibitor design is discussed.

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