Modulation of a Pre-existing Conformational Equilibrium Tunes Adenylate Kinase Activity

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• 作者：J枚rgen 脜d茅n ; Abhinav Verma ; Alexander Schug ; Magnus Wolf-Watz
• 刊名：The Journal of the American Chemical Society
• 出版年：2012
• 出版时间：October 10, 2012
• 年：2012
• 卷：134
• 期：40
• 页码：16562-16570
• 全文大小：484K
• 年卷期：v.134,no.40(October 10, 2012)
• ISSN：1520-5126

文摘

Structural plasticity is often required for distinct microscopic steps during enzymatic reaction cycles. Adenylate kinase from Escherichia coli (AK_eco) populates two major conformations in solution; the open (inactive) and closed (active) state, and the overall turnover rate is inversely proportional to the lifetime of the active conformation. Therefore, structural plasticity is intimately coupled to enzymatic turnover in AK_eco. Here, we probe the open to closed conformational equilibrium in the absence of bound substrate with NMR spectroscopy and molecular dynamics simulations. The conformational equilibrium in absence of substrate and, in turn, the turnover number can be modulated with mutational- and osmolyte-driven perturbations. Removal of one hydrogen bond between the ATP and AMP binding subdomains results in a population shift toward the open conformation and a resulting increase of k_cat. Addition of the osmolyte TMAO to AK_eco results in population shift toward the closed conformation and a significant reduction of k_cat. The Michaelis constants (K_M) scale with the change in k_cat, which follows from the influence of the population of the closed conformation for substrate binding affinity. Hence, k_cat and K_M are mutually dependent, and in the case of AK_eco, any perturbation that modulates k_cat is mirrored with a proportional response in K_M. Thus, our results demonstrate that the equilibrium constant of a pre-existing conformational equilibrium directly affects enzymatic catalysis. From an evolutionary perspective, our findings suggest that, for AK_eco, there exists ample flexibility to obtain a specificity constant (k_cat/K_M) that commensurate with the exerted cellular selective pressure.