Two-dimensional 1H-NMR studies of phospholipase-A2-inhibitor complexes bound to a micellar lipid-water interface.
One- and two-dimensional NMR studies were performed on the complexes of porcine pancreatic phospholipase A2 with substrate analogs bound to a micellar lipid-water interface of fully deuterated dodecylphosphocholine. The interactions between the inhibitor and the enzyme were localized by comparison of the two-dimensional NOE spectra recorded for the enzyme-inhibitor complex using both protonated and selectively deuterated inhibitors. These experiments led us to the following conclusions for the phospholipase-A2-micelle complex: (i) the 38-kDa phospholipase A2 complex gives NMR spectra with relatively narrow lines, which is indicative of high mobility of the enzyme; (ii) the residues Ala1, Trp3, Phe63 and Tyr69 located in the interface recognition site, as well as Phe22, Tyr75, Phe106 and Tyr111 are involved in the micelle-binding process; (iii) when present on the micelle, phospholipase A2 is stereospecific for the inhibitor binding; (iv) the inhibitor, (R)-dodecyl-2-aminohexanol-1-phosphoglycol, binds stoichiometrically to phospholipase A2 with high affinity (Kd less than or equal to 10 microM); (v) the inhibitor binds in the active site of the enzyme, which is evidenced by large chemical-shift differences for Phe5, Ile9, Phe22, His48, Tyr52 and Phe106; (vi) the acyl chain of the inhibitor makes hydrophobic contacts (less than 0.4 nm) near Phe5, Ile9, Phe22 and Phe106. Comparison of our results on the enzyme-inhibitor-micelle ternary complex with the crystal structure of the enzyme-inhibitor complex [Thunnissen, M. M. G. M., AB, E., Kalk, K. H., Drenth, J., Dijkstra, B. W., Kuipers, O. P., Dijkman, R., de Haas, G. H. & Verheij, H. M. (1990) Nature 347, 689-691] shows that the mode of inhibitor binding is similar.
Dekker
N
N
Center for Biomembranes and Lipid Enzymology, State University of Utrecht, Utrecht, The Netherlands.
Peters
A R
AR
Slotboom
A J
AJ
Boelens
R
R
Kaptein
R
R
Dijkman
R
R
de Haas
G
G
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008823:Micelles; D010179:Pancreas / Q000201:enzymology; D010741:Phospholipases A / Q000037:antagonists & inhibitors* / Q000737:chemistry; D054467:Phospholipases A2; D010743:Phospholipids / Q000494:pharmacology*; D010767:Phosphorylcholine / Q000031:analogs & derivatives; D011487:Protein Conformation; D013552:Swine; D014867:Water
D008823:Micelles; D010743:Phospholipids; D014867:Water; D010767:Phosphorylcholine; C070286:dodecyl-2-aminohexanol-1-phosphoglycol; C028810:dodecylphosphocholine; D006859:Hydrogen; D010741:Phospholipases A; D054467:Phospholipases A2