ABSTRACT: X-ray crystal structures of bovine pancreas prophospholipase A2 (proPLA2) inhibited by two amide-type inhibitors, [(R)-2-dodecanoyl-amino-1-hexanolphosphocholine (DAHPc) and (R)-2-dodecanoylamino-1-hexanolphosphoglycol (DAHPg)], were determined to R=0.208 and 0.215 using reflections with up to 2.1 Å resolution, respectively. Both complex crystals lacked defined electron densities for the prosequence of the N-terminal and for a loop region consisting of residues 65-70, retaining the disordered feature observed in free proPLA2 despite stabilization due to complex formation. The polar and nonpolar moieties of the amide-type inhibitors were located in the calcium-binding pocket and in the N-terminal α-helical hydrophobic region of the enzyme, respectively. As for the amide group of the inhibitor, which is lacking in the true substrate, a strong hydrogen bond was formed between the NH of the inhibitor and the unprotonated Nδ1 atom of His-48, resulting in the tight binding of the inhibitor to proPLA2, as well as to PLA2. The 20–30 times more potent inhibitory activity of DAHPg than DAHPc toward PLA2 could be explained by hydrogen bond formation between the glycol OH of DAHPg and the carbonyl O of Asp-49. The seven residues of the N-terminal prosequence of proPLA2, though disordered, block the access of a water molecule to Ala-1 of PLA2 or change the hydrogen-bonding property of Ala-1 α-amino group, resulting in breakage of the water-mediated hydrogen-bond network which is commonly formed in PLA2. The results of molecular dynamics (MD) calculation in an aqueous solution at 300 K indicate that this, rather than the close contact between the prosequence and the residues 65-70 loop region, is the main reason why the latter region becomes flexible in proPLA2, compared with in PLA2.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology.