Substrate-induced product-release mechanism of lipocalin-type prostaglandin D synthase.

Prostaglandin D (PGD), an endogenous somnogen, is a unique PG that is secreted into the cerebrospinal fluid. PGD is a relatively fragile molecule and should be transported to receptors localized in the basal forebrain without degradation. However, it remains unclear how PGD is stably carried to such remote receptors. Here, we demonstrate that the PGD-synthesizing enzyme, Lipocalin-type prostaglandin D synthase (L-PGDS), binds not only its substrate PGH but also its product PGD at two distinct binding sites for both ligands. This behaviour implys its PGD carrier function. Nevertheless, since the high affinity (K = ∼0.6 μM) of PGD in the catalytic binding site is comparable to that of PGH, it may act as a competitive inhibitor, while our binding assay exhibits only weak inhibition (K = 189 μM) of the catalytic reaction. To clarify this enigmatic behavior, we determined the solution structure of L-PGDS bound to one substrate analog by NMR and compared it with the two structures: one in the apo form and the other in substrate analogue complex with 1:2 stoichiometry. The structural comparisons showed clearly that open or closed forms of loops at the entrance of ligand binding cavity are regulated by substrate binding to two sites, and that the binding to a second non-catalytic binding site, which apparently substrate concentration dependent, induces opening of the cavity that releases the product. From these results, we propose that L-PGDS is a unique enzyme having a carrier function and a substrate-induced product-release mechanism.