Tafazzin catalyzes transacylation and hydrolysis with inverse acyl specificities

Tafazzin is a phospholipid-lysophospholipid acyltransferase that remodels fatty acids of mitochondrial phospholipids. To study the tafazzin mechanism, we expressed and purified the enzyme, measured its kinetics with different pairs of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) species, and simulated the reactions in a mathematical model based on the mass-action law. Variations in chain length and PC:LPC ratio showed that tafazzin reacts with micelles but not with bilayers. In the presence of micellar substrates (LPC + short-chain PC), transacylation rates measured at different substrate ratios suggested similar affinities for PC and LPC. Surprisingly, tafazzin catalyzed both transacylation and hydrolysis of fatty acids, the former preferring oleoyl over palmitoyl and the latter preferring palmitoyl over oleoyl groups. To study the remodeling of cardiolipin, we measured the reaction of trilinoleoyl-monolyso-cardiolipin with palmitoyl-oleoyl-PC and simulated it in a mathematical model. Simulations predicted and measurements confirmed a large number of bidirectional transacylations between the initial substrates, between substrates and products, and between different products. This led to a gradual diversification of the lipid composition from 2 to more than 15 species. Discrepancies between observed and simulated compositions demonstrated transacylation preference for oleoyl over palmitoyl groups. The data are consistent with a bidirectional ping-pong mechanism that operates through a single low-affinity binding site and suggest that transacylation specificity can at least in part be explained by selective loss of fatty acids through hydrolysis.