The binding of amide substrate analogues to phospholipase A2. Studies by 13C-nuclear-magnetic-resonance and infrared spectroscopy

摘要

p(R)-(2-dodecanamidoisohexyl)phosphocholine (DAHPC), labelled with 13C at the amide carbonyl group, has been synthesized and its binding to bovine pancreatic phospholipase A2 (PLA2) studied by n.m.r. and i.r. spectroscopy. Two-dimensional 1H-n.m.r. spectra show that, in the presence of Ca2+, DAHPC binds to the active site of the enzyme in a similar manner to other phospholipid amide substrate analogues. The environment of the labelled carbonyl group has been investigated by a combination of 13C n.m.r. and difference-Fourier-transform i.r. spectroscopy. The carbonyl resonance shifts 3 p.p.m. downfield on the binding of DAHPC to PLA2. The carbonyl absorption frequency decreases by 14-18 cm-1, accompanied by a marked sharpening of the absorption band. These results indicate that the carbonyl bond undergoes significant polarization in the enzyme-ligand complex, facilitated by the enzyme-bound Ca2+ ion. This suggests that ground-state strain is likely to promote catalysis in the case of substrate binding. Simple calculations, based on the i.r. data, indicate that the carbonyl bond is weakened by 5-9 kJ.mol-1. This is the first report of observation of the amide vibration of a bound ligand against the strong background of protein amide vibrations./p