title: X-ray crystal structure determination and molecular dynamics simulation of prophospholipase A2 inhibited by amide-type substrate analogues.
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 A 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 alpha-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 N(delta1) 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 alpha-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.
authors.affiliation: Osaka University of Pharmaceutical Sciences, Takatsuki, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Tomoo
authors.forename: A
authors.initials: A
authors.lastname: Yamane
authors.forename: T
authors.initials: T
authors.lastname: Ishida
authors.forename: S
authors.initials: S
authors.lastname: Fujii
authors.forename: K
authors.initials: K
authors.lastname: Ikeda
authors.forename: S
authors.initials: S
authors.lastname: Iwama
authors.forename: S
authors.initials: S
authors.lastname: Katsumura
authors.forename: S
authors.initials: S
authors.lastname: Sumiya
authors.forename: H
authors.initials: H
authors.lastname: Miyagawa
authors.forename: K
authors.initials: K
authors.lastname: Kitamura
mesh_terms: D000818:Animals; D002384:Catalysis; D004792:Enzyme Precursors; D007850:Lauric Acids / Q000494:pharmacology; D015394:Molecular Structure; D009943:Organophosphorus Compounds / Q000494:pharmacology; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000737:chemistry*; D054467:Phospholipases A2; D011498:Protein Precursors / Q000037:antagonists & inhibitors / Q000737:chemistry*; D013552:Swine; D013816:Thermodynamics; D014961:X-Ray Diffraction
chemical_list: D004792:Enzyme Precursors; D007850:Lauric Acids; D009943:Organophosphorus Compounds; D011498:Protein Precursors; C093914:2-dodecanoylamino-1-hexanolphosphocholine; C066179:2-dodecanoylamino-1-hexanolphosphoglycol; D010741:Phospholipases A; D054467:Phospholipases A2; C085333:prophospholipase A2