Date of Award

Fall 2022

Degree Type


Degree Name

Master of Science (MS)




During affinity maturation, antibodies acquire point mutations that lead to enhanced binding strength to a particular ligand while simultaneously becoming more conformationally restricted as they become increasingly complementary to their ligand’s electrostatic geometry. This property has catalytic potential in transition-state (TS) stabilization whereby kinetic barriers (i.e., the energetic gap between the substrate and its TS) are reduced and reaction rates are enhanced. This basic mechanism is thought to underlie the majority of the rate enhancement observed for enzymes. A key integration of antibody binding specificity and strength with enzymatic catalytic strategy is the development of catalytic antibodies (“abzymes”). Abzymes are designed to bind a single, ideally rate-limiting TS of a given organic reaction. While abzymes are an impressive verification of enzymatic TS stabilization as a catalytic strategy, abzymes fall short of enzymatic rates. A largely overlooked explanation for this pertains to conformational heterogeneity in abzymes compared to enzymes. Abzymes are stiff lock-and-key binders with little to no conformational arrangements, as their function demands rigid specificity. Enzymes, by contrast, are flexible proteins subject to directed conformational shifts that are mechanochemically coupled to the substrate. In the present work, we attempted to restore conformational heterogeneity to a model abzyme to examine its effect upon catalysis. We do this using the natural maturation process of antibodies, somatic hypermutation. We identified residues potentially contributing to rigidity both via somatic hypermutation and through cross-reference to a database of common mutations we compiled. We evaluate catalytic performance with steady-state kinetics, and flexibility with spectroscopic and computational techniques. We observed dramatic falls in turnover rates, but with improvements in the Michealis constant (KM). We interpret this general trend as improved binding to substrate as a result of enhanced conformational sampling, but while also raising the barrier to the TS thus lowering turnover rates. We found only weak relationships between catalytic parameters and proxies for flexibility; however, indicating either our proxies failed to capture significant changes in conformational distributions or that conformational heterogeneity is unrelated to the loss of catalytic capacity in our selected mutations.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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