Structural importance of the acyl group in substrate specificity of purified bovine lysophospholipase D.

The structural importance of the acyl group in lysophosphatidylcholine (LPC) as substrate of purified bovine lysophospholipase D (lysoPLD) was investigated. Among LPCs with saturated acyl chains, the K(m) value decreased according to the length of the acyl chain (C12-C16) up to the palmitoyl group, while the V(m) value showed no remarkable change. But, the extension of the acyl size to C18, as observed with 1-stearoyl LPC (K(m), 8.5 mM), rather resulted in a remarkable increase in the K(m) value. Meanwhile, the introduction of one double bond in the C18 saturated acyl chain led to a remarkable reduction in the K(m) value, as observed with 1-oleoyl LPC (K(m), 0.48 mM). Furthermore, 1-linoleoyl LPC (K(m), 56 microM) with two double bonds exhibited a smaller K(m) value than 1-oleoyl LPC, suggesting that the unsaturation degree might be important in augmenting the binding affinity of LPCs. A similar phenomenon was also observed with 1-arachidonoly LPC (K(m), 79 microM) or 1-docosahexaenoyl LPC (K(m), 36 microM). Overall, the order of catalytic efficiency (V(m)/K(m) value) of those LPCs seemed to be affected by the K(m) value rather than the V(m) value, which differed by at most threefold among LPC derivatives. Next, the introduction of a hydroperoxide group into 1-linoleoyl-LPC or 1-arachidonoyl LPC led to a further reduction in K(m) values (1-hydroperoxylinoleoyl LPC, 26 microM; 1-hydroperoxyarachidonoyl LPC, 33 microM), accompanied by a further increase in the V(m)/K(m) values. Additionally, phosphatidylcholines (PCs) with an oxidized acyl chain at sn-2 position were found to be efficient as 1-palmitoyl LPC as substrates of lysoPLD. Taken together, the catalytic efficiency of LPCs or oxidized PCs as substrates of lysoPLD seems to be determined by the property of the acyl chain, length of the acyl chain, unsaturation degree and oxidation status.