Tawfik D S, Lindner A B, Chap R, Eshhar Z, Green B S
Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel.
Eur J Biochem. 1997 Mar 1;244(2):619-26. doi: 10.1111/j.1432-1033.1997.00619.x.
A number of monoclonal antibodies elicited against a nitrobenzyl (Nbzl)-phosphonate transition-state analogue (TSA), and which were selected for the hydrolysis of the corresponding Nbzl-ester, were also found to catalyze the hydrolysis of the analogous p-nitrophenyl(Np) ester with notable efficiency and specificity. The activity towards the Np-ester is higher in terms of rates (k(cat); as expected from the higher intrinsic reactivity of Np-esters); however, the rate acceleration (k(cat)/k(uncat)) is close to or lower than that observed with the Nbzl-ester. Unexpectedly, the affinity to the Np-ester substrate (1/K(M)) and therefore k(cat)/K(M) are significantly higher. The best example is antibody D2.4 having a k(cat)/K(M) value of 64 s(-1) x M(-1) with the Nbzl-ester and 9400 s(-1) x M(-1) with the Np-ester. Moreover, due to a lower product inhibition by p-nitrophenol relative to p-nitrobenzyl alcohol, these antibodies exhibit more than 1000 turnovers with the Np-ester. The differential affinity of these antibodies to the Nbzl-phosphonate TSA versus the Nbzl-ester substrate (K(S)/K(TSA) or K(M)/K(i)) correlates well with the observed rate enhancement (k(cat)/k(uncat)). For the Np-ester, however, stabilisation of the transition state (as reflected by K(S)/K(TSA) and by the catalytic proficiencies, k(cat)/K(M)/k(uncat)) does not fully account for the catalytic power (k(cat)/k(uncat)), indicating a more complex catalytic mechanism than simply transition-state stabilization. A comparison of the kinetic parameters of D2.4 with other Np-ester-hydrolyzing antibodies raised against Np-phosphonate haptens emphasizes the marked advantage of this antibody which was elicited against an Nbzl-phosphonate hapten. These results appear to be general: anti-(Nbzl-phosphonate TSA) antibodies obtained from other mouse strains and using different immunization protocols are also efficient Np-esterases. They demonstrate the use of an expanded TSA-hapten, where a spacer (a methylene group) mimics bonds that are partially cleaved in the transition state of the catalyzed reaction.
一些针对硝基苄基（Nbzl）-膦酸酯过渡态类似物（TSA）产生的单克隆抗体，这些抗体因能催化相应Nbzl酯的水解而被筛选出来，结果发现它们也能高效且特异地催化类似的对硝基苯基（Np）酯的水解。就反应速率（k(cat)）而言，这些抗体对Np酯的活性更高（正如Np酯更高的内在反应活性所预期的那样）；然而，速率加速（k(cat)/k(uncat)）接近或低于Nbzl酯的情况。出乎意料的是，这些抗体对Np酯底物的亲和力（1/K(M)）以及因此的k(cat)/K(M)显著更高。最好的例子是抗体D2.4，它对Nbzl酯的k(cat)/K(M)值为64 s(-1)×M(-1)，对Np酯的k(cat)/K(M)值为9400 s(-1)×M(-1)。此外，由于对硝基苯酚相对于对硝基苄醇的产物抑制作用更低，这些抗体对Np酯能表现出超过1000次的周转。这些抗体对Nbzl-膦酸酯TSA与Nbzl酯底物的差异亲和力（K(S)/K(TSA)或K(M)/K(i)）与观察到的速率增强（k(cat)/k(uncat)）密切相关。然而，对于Np酯，过渡态的稳定化（由K(S)/K(TSA)以及催化效率k(cat)/K(M)/k(uncat)反映）并不能完全解释催化能力（k(cat)/k(uncat)），这表明催化机制比简单的过渡态稳定化更为复杂。将D2.4的动力学参数与针对Np-膦酸酯半抗原产生的其他Np酯水解抗体进行比较，突出了这种针对Nbzl-膦酸酯半抗原产生的抗体的显著优势。这些结果似乎具有普遍性：从其他小鼠品系获得的、采用不同免疫方案的抗（Nbzl-膦酸酯TSA）抗体也是高效的Np酯酶。它们证明了扩展TSA-半抗原的应用，其中一个间隔基团（一个亚甲基）模拟了在催化反应过渡态中部分断裂的键。
