Angeles T S, Smith R G, Darsley M J, Sugasawara R, Sanchez R I, Kenten J, Schultz P G, Martin M T
IGEN, Inc., Rockville, Maryland 20852.
Biochemistry. 1993 Nov 16;32(45):12128-35. doi: 10.1021/bi00096a025.
Mechanistic and structural comparisons of five catalytic monoclonal antibodies generated from the same hybridoma fusion indicated that all five hydrolyze phenyl acetate by subtle variations of the same mechanism. All of the antibodies showed a pre-steady-state multi-turnover burst in which kcat and Km declined but kcat/Km did not change. The burst of one of the antibodies, 20G9, has previously been found to result from inhibition by the product, phenol. Although all of the antibodies showed the burst, their individual values for kcat, Km, and hapten Ki differed substantially. Three of the antibodies that were investigated for the effect of pH on kcat showed an acid limb pK of 9.5-9.6. Substrate inhibition was seen in four of the five antibodies. Variable region nucleotide sequencing of the heavy and light chains confirmed that all five antibodies were structurally similar and also revealed several potentially critical tyrosines. Despite their structural similarities, analysis of their sequences suggested that the antibodies are products of distinct, independent rearrangements of immunoglobulin gene segments that took place in different progenitor B cells. A plot of Ki for hapten inhibition vs Km/kcat for substrate hydrolysis for the mechanistically related antibodies ("isoabzymes") gave a linear relationship suggesting a catalytic role for transition-state complementarity. Taken together with previous work [Martin et al. (1991) Biochemistry 30, 9757-9761], the data conform to a mechanism in which the antibodies exploit both transition-state complementarity and an acyl-tyrosyl intermediate during phenyl acetate hydrolysis.
对由同一杂交瘤融合产生的五种催化单克隆抗体进行的机制和结构比较表明，所有这五种抗体通过相同机制的细微变化来水解苯乙酸。所有抗体均显示出预稳态多周转爆发，其中kcat和Km下降，但kcat/Km不变。先前已发现其中一种抗体20G9的爆发是由产物苯酚的抑制作用导致的。尽管所有抗体都显示出爆发，但它们各自的kcat、Km和半抗原Ki值有很大差异。对其中三种抗体研究了pH对kcat的影响，结果显示酸侧链pK为9.5 - 9.6。在五种抗体中的四种中观察到了底物抑制。重链和轻链的可变区核苷酸测序证实，所有五种抗体在结构上相似，同时也揭示了几个潜在的关键酪氨酸。尽管它们在结构上相似，但对其序列的分析表明，这些抗体是免疫球蛋白基因片段在不同祖B细胞中发生的不同、独立重排的产物。对于机制相关的抗体（“同功抗体”），半抗原抑制的Ki与底物水解的Km/kcat作图得到线性关系，表明过渡态互补具有催化作用。结合先前的工作[Martin等人（1991年）《生物化学》30, 9757 - 9761]，这些数据符合一种机制，即抗体在苯乙酸水解过程中利用过渡态互补和酰基 - 酪氨酰中间体。
