Catalytic Proficiency of Ubiquitin Conjugation Enzymes: Balancing pKa Suppression, Entropy, and Electrostatics

Biological organisms orchestrate coordinated responses to external stimuli through temporal fluctuations in protein−protein interaction networks using molecular mechanisms such as the synthesis and recognition of polyubiquitin (polyUb) chains on signaling adaptor proteins. One of the pivotal chemical steps in ubiquitination involves reaction of a lysine amino group with a thioester group on an activated E2, or ubiquitin conjugation enzyme, to form an amide bond between Ub and a target protein. In this study, we demonstrate a nominal 14-fold range for the rate of the chemical step, kcat, catalyzed by different E2 enzymes using non-steady-state, single-turnover assays. However, the observed range for kcat is as large as ∼100-fold for steady-state, single-turnover assays. Biochemical assays were used in combination with measurement of the underlying protein−protein interaction kinetics using NMR line-shape and ZZ-exchange analyses to determine the rate of polyUb chain synthesis catalyzed by the heterodimeric...