Slaich P K, Primrose W U, Robinson D H, Wharton C W, White A J, Drabble K, Roberts G C
Department of Biochemistry and Biological N.M.R. Centre, University of Leicester, U.K.
Biochem J. 1992 Nov 15;288 ( Pt 1)(Pt 1):167-73. doi: 10.1042/bj2880167.
(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.
已合成了在酰胺羰基处标记有¹³C的(R)-(2-十二烷酰胺基异己基)磷酰胆碱(DAHPC)，并通过核磁共振和红外光谱研究了其与牛胰磷脂酶A2(PLA2)的结合情况。二维¹H-核磁共振谱表明，在Ca²⁺存在下，DAHPC以与其他磷脂酰胺底物类似物相似的方式结合到酶的活性位点。通过¹³C核磁共振和差示傅里叶变换红外光谱相结合的方法研究了标记羰基的环境。DAHPC与PLA2结合时，羰基共振向低场移动3 ppm。羰基吸收频率降低14 - 18 cm⁻¹，同时吸收带明显变锐。这些结果表明，在酶-配体复合物中，羰基键发生了显著极化，这是由与酶结合的Ca²⁺离子促成的。这表明在底物结合的情况下，基态应变可能促进催化作用。基于红外数据的简单计算表明，羰基键减弱了5 - 9 kJ·mol⁻¹。这是首次在蛋白质酰胺振动的强背景下观察到结合配体的酰胺振动的报道。
