Lombardo D, Fanni T, Plückthun A, Dennis E A
J Biol Chem. 1986 Sep 5;261(25):11663-6.
H2(18)O isotope exchange into specifically 13C-labeled substrate was used to obtain information on the rate-limiting step in the action of the phospholipase A2 from the venom of the Indian cobra (Naja naja naja). Incorporation of 18O was detected by the effect of 18O on 13C chemical shifts in 13C NMR. The enzymatic hydrolysis of a micellar phosphatidylcholine analogue of platelet-activating factor 1-alkyl-2-[1-13C]lauroyl-sn-glycero-3-phosphorylcholine proceeds by an O-acyl cleavage of the sn-2 ester bond. The reaction was examined for simultaneous 18O incorporation into the substrate. No exchange was found, suggesting that the hydrolytic step is not followed by a higher energy transition state and that it or a step before it appears to be rate-limiting. Previous experiments on phosphatidylethanolamine activation indicate that kcat is altered but that the km remains the same upon activation, suggesting that the binding steps occurring before the hydrolytic step are not affected. This strongly suggests that the hydrolytic step is in fact the rate-limiting step under these conditions. The 13C, 18O NMR technique should be generally applicable to mechanistic questions of this type.
利用H₂(¹⁸)O同位素与特定的¹³C标记底物进行交换，以获取有关印度眼镜蛇（Naja naja naja）毒液中磷脂酶A₂作用限速步骤的信息。通过¹³C NMR中¹⁸O对¹³C化学位移的影响来检测¹⁸O的掺入情况。血小板活化因子1-烷基-2-[1-¹³C]月桂酰-sn-甘油-3-磷酸胆碱的胶束磷脂酰胆碱类似物的酶促水解通过sn-2酯键的O-酰基裂解进行。研究了该反应中¹⁸O同时掺入底物的情况。未发现交换现象，这表明水解步骤之后没有更高能量的过渡态，并且水解步骤或其之前的某个步骤似乎是限速步骤。先前关于磷脂酰乙醇胺活化的实验表明，活化后kcat发生改变，但km保持不变，这表明水解步骤之前发生的结合步骤不受影响。这有力地表明，在这些条件下，水解步骤实际上是限速步骤。¹³C、¹⁸O NMR技术应普遍适用于此类机理问题。
