X-RAY CRYSTAL-STRUCTURE DETERMINATION AND MOLECULAR-DYNAMICS SIMULATION OF PROPHOSPHOLIPASE A(2) INHIBITED BY AMIDE-TYPE SUBSTRATE-ANALOGS

S-ray crystal structures of bovine pancreas prophospholipase A(2) (Fro PLA(2)) inhibited by two amide-type inhibitors, [(R)-2-dodesanoyl-amin o-1-hexanolphosphocholine (DAHPc) and (R)-2-dodecanoylamino-1-hexanolp hosphoglycol (DAHPg)], were determined to R = 0.208 and 0.215 using re flections with up to 2.1 Angstrom revolution, respectively, Both compl ex crystals lacked defined electron densities for the prosequence of t he N-terminal and for a loop region consisting of residues 65-70, reta ining the disordered feature observed in free proPLA, despite stabiliz ation 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 cu-helical hydrophobic region of the enzyme, respect ively. As for the amide group of the inhibitor, which is lacking in th e true substrate, a strong hydrogen bond was formed between the NH of the inhibitor and the unprotonated N-delta 1 atom of His-48, resulting in the tight binding of the inhibitor to proPLA(2). as well as to PLA (2). The 20-30 times more potent inhibitory activity of DAHPg than DAH Pc toward PLA(2) could be explained by hydrogen bond formation between the glycol OH of DAHPg and the carbonyl O of Asp-49. The seven residu es of the N-terminal prosequence of proPLA(2), though disordered, bloc k the access of a water molecule to Ala-l of PLA(2) or change the hydr ogen-bonding property of Ala-l cu-amino group, resulting in breakage o f the water-mediated hydrogen-bond network which is commonly formed in PLA(2). The results of molecular dynamics (MD) calculation in an aque ous solution at 300 K indicate that this, rather. than the close conta ct between the prosequence and the residues 65-70 loop region. is the main reason why the latter region becomes flexible in proPLA(2), compa red with in P6A(2).