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

Abstract

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, k(cat), catalyzed by different E2 enzymes using non-steady-state, single-turnover assays. However, the observed range for k(cat) 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 E2 enzyme Ubc13-Mms2. Modest variations in substrate affinity and k(cat) can achieve functional diversity in E2 mechanism, thereby influencing the biological outcomes of polyubiquitination. E2 enzymes achieve reaction rate enhancements through electrostatic effects such as suppression of substrate lysine pK(a) and stabilization of transition states by the preorganized, polar enzyme active site as well as the entropic effects of binding. Importantly, modestly proficient enzymes such as E2s maintain the ability to tune reaction rates; this may confer a biological advantage for achieving specificity in the diverse cellular roles for which these enzymes are involved.

Exploring the RING-Catalyzed Ubiquitin Transfer Mechanism by MD and QM/MM Calculations

“…To be consistent with our calculations, mutations of the H75 and N77 on E2 led to decrease in reactivity [40], [42], [73], [74]. These observations also show that electrostatic complementarity is one of the key functions for E2, which agrees with former investigation [43]. From the curve plotted in Figure 2B, the energy barrier needed for nucleophilic attack is 4.23 kcal/mol.…”

RYBP Is a K63-Ubiquitin-Chain-Binding Protein that Inhibits Homologous Recombination Repair

“…RAP80, which also binds to K63 ubiquitin, contains two ubiquitin-interacting motif (UIM) domains, which allow it to bind with high affinity (Markin et al, 2010b;Sims and Cohen, 2009). Interestingly, the affinity of the RYBP-NZF domain for monoubiquitin (K D $220 mM) is higher than that of the individual RAP80 UIM domains ($500-900 mM) (Markin et al, 2010a). The affinity of both proteins for K63-diubiquitin is similar ($20-80 mM).…”

Stochastic Gate Dynamics Regulate the Catalytic Activity of Ubiquitination Enzymes

“…Thus, loop dynamics provide a thin layer of regulation on top of a large catalytic advantage. 9 Yet, defects within this regulatory shell can have profound biological consequences, considering that Ubc13 is the only E2 dedicated to synthesizing Lys 63-linked polyUb chains. Ubc13-D118G and A122G show respective 2-and 40-fold reductions for the in vitro turnover rate (k cat,obs ) for attachment of the second Ub during synthesis of Lys 63-linked polyUb chains.…”

“…In water, k cat,2 is ∼0.3 M −2 s −1 for aminolysis reactions of small molecule model compounds; 37 this 10 8 fold increase in the rate constant underscores the catalytic prowess of E2 enzymes; the cost of catalyzing biologically relevant aminolysis is paid for in large part by the structure and dynamics of the E2 enzyme. 9 Unlike hydrolysis, 15 N NMR relaxation parameters (600 MHz) for the stochastic gate from the E2 Ubc13 and gate mutants. For L121G, the contribution to R 2 from micro-to millisecond time scale dynamics (R ex ) was estimated as previously described 52 and subtracted from the experimental R 2 value.…”