Detection of a catalytic antibody species acylated at the active site by electrospray mass spectrometry.
Detection of a catalytic antibody species acylated at the active site by electrospray mass spectrometry.
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通过电喷雾质谱法检测活性位点酰化的催化抗体种类。
DOI：
10.1021/bi00003a002
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Benkovic,SJ
中科院分区：
文献类型：
--
作者：
Krebs,JF;Siuzdak,G;Dyson,HJ;Stewart,JD;Benkovic,SJ
1994年11月28日接收的修订版Mandarin pt ®摘要：使用电喷雾（离子喷雾）质谱法检查催化抗体Fv片段和酯底物之间的化学相互作用。在将对硝基苯基酯底物添加至抗体片段后，在电喷雾光谱中清楚地观察到代表约8%的总Fv浓度的抗体片段种类。观察到的Fv片段的分子量增加对应于底物的酰基的质量。通过将抗体片段与半抗原抑制剂预孵育来阻断酰基-Fv种类的形成，这表明酰基连接涉及抗体活性位点中的残基。当使用相应的对氯苯酯底物时，未观察到酰基-Fv物质，表明该物质的水平取决于底物的离去基团。酰化的物种没有观察到的定点突变缺乏催化活性，组氨酸L91 Gin。目前的结果与Fv片段结构的建模研究一致，并且为先前针对该抗体提出的多步动力学机制提供了强有力的证实证据。NPN 43 C9是催化芳香族苯胺和一系列相关苯酯水解的催化抗体（Janda等人，1988年）。该抗体由膦酰胺半抗原（1）诱导，该半抗原类似于酯（2a，2b）或苯胺（2c）水解的四面体过渡态。
Revised Manuscript Received November 28, 1994® abstract: The chemical interactions between a catalytic antibody Fv fragment and ester substrates were examined using pneumatically assisted electrospray (ion spray) mass spectrometry. Upon addition of the p-nitrophenyl ester substrate to the antibody fragment, an antibody fragment species that represents approximately 8% of the total Fv concentration is clearly observed in the electrospray spectrum. The observed increase in molecular weight of the Fv fragment corresponds to the mass of the acyl group of the substrate. Formation of the acyl—Fv species is blockedby preincubation of the antibody fragment with hapten inhibitor, suggesting that the acyl linkage involves a residue in the active site of the antibody. The acyl—Fv species is not observed when the corresponding^-chlorophenyl ester substrate is used, indicating that the level of this species is dependent on the leaving group of the substrate. The acylated species is not observed for a site-directed mutant lacking catalytic activity, His L91 Gin. The present results are consistent with modeling studies of the structure of the Fv fragment and provide strong confirmatory evidence for the multistepkinetic mechanism previously proposed for this antibody.NPN43C9 is a catalytic antibody which catalyzesthe hydrolysis of an aromatic anilide and a series of related phenyl esters (Janda et al., 1988). This antibody was induced by a phosphonamidate hapten (1) which resembles the tetrahedral transition state for ester (2a, 2b) or anilide (2c) hydrolysis.