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

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• 作者：Craig J. Markin ; Linda F. Saltibus ; Melissa J. Kean ; Ryan T. McKay ; Wei Xiao ; Leo Spyracopoulos
• 刊名：Journal of the American Chemical Society
• 出版年：2010
• 出版时间：December 22, 2010
• 年：2010
• 卷：132
• 期：50
• 页码：17775-17786
• 全文大小：421K
• 年卷期：v.132,no.50(December 22, 2010)
• ISSN：1520-5126

文摘

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.