The binding of amide substrate analogues to phospholipase A2. Studies by 13C-nuclear-magnetic-resonance and infrared spectroscopy.
(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.
Slaich
P K
PK
Department of Biochemistry and Biological N.M.R. Centre, University of Leicester, U.K.
Primrose
W U
WU
Robinson
D H
DH
Wharton
C W
CW
White
A J
AJ
Drabble
K
K
Roberts
G C
GC
D000818:Animals; D001665:Binding Sites; D002247:Carbon Isotopes; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D005583:Fourier Analysis; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D010179:Pancreas / Q000201:enzymology; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D054467:Phospholipases A2; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D013055:Spectrophotometry, Infrared; D013816:Thermodynamics
D002247:Carbon Isotopes; D010767:Phosphorylcholine; D010741:Phospholipases A; D054467:Phospholipases A2