Esterolytic antibodies as mechanistic and structural models of hydrolases-a quantitative analysis.
Understanding enzymes quantitatively and mimicking their remarkable catalytic efficiency is a paramount challenge. Here, we applied esterolytic antibodies (the D-Abs) to dissect and quantify individual elements of enzymatic catalysis such as transition state (TS) stabilization, nucleophilic reactivity and conformational changes. Kinetic and mutagenic analysis of the D-Abs were combined with existing structural evidence to show that catalysis by the D-Abs is driven primarily by stabilization of the tetrahedral oxyanionic intermediate of ester hydrolysis formed by the nucleophilic attack of an exogenous (solution) hydroxide anion. The side-chain of TyrH100d is shown to be the main H-bond donor of the D-Abs oxyanion hole. The pH-rate and pH-binding profiles indicate that the strength of this H-bond increases dramatically as the neutral substrate develops into the oxyanionic TS, resulting in TS stabilization of 5-7 kcal/mol, which is comparable to oxyanionic TS stabilization in serine hydrolases. We show that the rate of the exogenous (intermolecular) nucleophilic attack can be enhanced by 2000-fold by replacing the hydroxide nucleophile with peroxide, an alpha-nucleophile that is much more reactive than hydroxide. In the presence of peroxide, the rate saturates (k(cat)(max)) at 6 s(-1). This rate-ceiling appears to be dictated by the rate of the induced-fit conformational rearrangement leading to the active antibody-TS complex. The selective usage of negatively charged exogenous nucleophiles by the D-Abs led to the identification of a positively charged channel. Imprinted by the negatively-charged TS-analogue against which these antibodies were elicited, this channel presumably directs the nucleophile to the antibody-bound substrate. Our findings are discussed in comparison with serine esterases and, in particular, with cocaine esterase (cocE), which possesses a tyrosine based oxyanion hole.
Lindner
Ariel B
AB
Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel.
Kim
Se Ho
SH
Schindler
Dan G
DG
Eshhar
Zelig
Z
Tawfik
Dan S
DS
D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000941:Antigens / Q000737:chemistry; D002384:Catalysis; D004795:Enzyme Stability; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010545:Peroxides / Q000494:pharmacology; D011487:Protein Conformation; D013379:Substrate Specificity; D013816:Thermodynamics
D000906:Antibodies; D000941:Antigens; D010545:Peroxides; D006867:Hydrolases