Bifunctional Catalysis of Proton Transfer at an Antibody Active Site

The structure of antibody 13G5, which catalyzes the conversion of unactivated benzisoxazoles to the corresponding salicylonitriles, was determined at 2.65 Å resolution in the absence of ligand. It shows that the 2-aminobenzimidazolium derivative originally used for immunization was successful in templating an active site containing multiple functional groups. Thus, the side chains of three polar residues, AspH35, HisH95, and GluL34, project into an otherwise hydrophobic cavity. Site-directed mutagenesis and subsequent kinetic analysis identified AspH35 as the likely catalytic base that initiates proton abstraction. HisH95 appears to activate the base, whereas GluL34 may stabilize the incipient phenolate anion in the transition state at acidic pH. Introduction of alanine at position L34 unexpectedly boosts the activity of the catalyst substantially and broadens its pH optimum, perhaps because ordered water molecules are able to assume the role of proton donor over a wider range of conditions than the original glutamic acid. The aspartate base in the GluL34Ala variant provides an astonishing 109-fold rate advantage over the nonenzymatic reaction with acetate as base and exhibits an effective molarity of >106 M. These large effects illustrate the utility of bifunctional catalysis in an antibody active site for promoting reactions with unactivated substrates.

催化未活化苯并异噁唑转化为对应水杨腈的抗体13G5，在无配体条件下以2.65埃（Å）的分辨率解析了其晶体结构。结构分析显示，最初用于免疫的2-苯并咪唑鎓衍生物（2-aminobenzimidazolium derivative）成功模板化出一个带有多个官能团的活性位点。具体而言，三个极性残基——重链35位天冬氨酸（AspH35）、重链95位组氨酸（HisH95）以及轻链34位谷氨酸（GluL34）——的侧链伸入原本的疏水空腔中。通过定点诱变及后续动力学分析，确定AspH35是引发质子夺取的潜在催化碱。HisH95似乎负责活化该催化碱，而GluL34可能在酸性pH条件下稳定过渡态中初生的酚酸根阴离子。令人意外的是，将轻链34位的谷氨酸突变为丙氨酸（GluL34Ala）后，该催化剂的活性大幅提升且最适pH范围变宽，这可能是因为相较于原始的谷氨酸残基，有序水分子能够在更广泛的pH条件下充当质子供体。相较于以乙酸盐作为碱的非酶促反应，GluL34Ala突变体中的天冬氨酸催化碱可带来高达10^9倍的速率优势，且有效摩尔浓度超过10^6 M。这些显著的效应证明了在抗体活性位点中采用双功能催化，可有效促进未活化底物参与的化学反应。