Linkage between dynamics and catalysis in a thermophilic-mesophilic enzyme pair

Abstract

A fundamental question is how enzymes can accelerate chemical reactions. Catalysis is not only defined by actual chemical steps, but also by enzyme structure and dynamics. To investigate the role of protein dynamics in enzymatic turnover, we measured residue-specific protein dynamics in hyperthermophilic and mesophilic homologs of adenylate kinase during catalysis. A dynamic process, the opening of the nucleotide-binding lids, was found to be rate-limiting for both enzymes as measured by NMR relaxation. Moreover, we found that the reduced catalytic activity of the hyperthermophilic enzyme at ambient temperatures is caused solely by a slower lid-opening rate. This comparative and quantitative study of activity, structure and dynamics revealed a close link between protein dynamics and catalytic turnover.

Urea-Dependent Adenylate Kinase Activation following Redistribution of Structural States

“…[4][5][6] (denoted ''model 1'') well, yielding DG 0 fold and m fold values of 13.7 kJ mol À1 and 7.2 kJ mol À1 M À1 , respectively (Fig. 2).…”

“…In protein folding, the m value is proportional to the difference in SAS area between the folded ground state and transition state for the folding/unfolding process. Because the catalytic rate of Adk is limited by slow opening of the substrate-binding domains in the presence of bound nucleotides (5,6), the m act value will be proportional to the difference in SAS area between the closed state and the transition state associated with the opening reaction.…”

“…In the case of Adk, the compensatory effect is dependent on the limitation of catalytic activity by opening of substrate-binding domains (k cat ¼ k open in Scheme 1) in the presence of bound nucleotides (5 (Fig. 9).…”

“…Similarly, enzymatic activity is often dependent on conformational changes during their reaction cycles, and it has been established for a few enzymes that the dynamic interconversions between structural microstates are rate-limiting for turnover of substrate molecules (3,4). For instance, the rate of the reaction catalyzed by Escherichia coli adenylate kinase (Adk), is limited by slow reopening of substrate-binding subdomains in the presence of bound nucleotides (5)(6)(7), as illustrated in the following minimal reaction scheme. Here, E refers to Adk, S to substrate, P to product, and superscripts open and closed to Adk in open and closed states (Fig.…”

Characterizing Active Site Conformational Heterogeneity along the Trajectory of an Enzymatic Phosphoryl Transfer Reaction

“…Crystal structures of NMP kinases in several states have shown substantial conformational changes upon substrate binding and revealed the enthalpic stabilization along the reaction coordinate,6 including the transition state of the chemical phosphoryl transfer step (Figure 1). 7 Also, backbone dynamics of the NMP kinase adenylate kinase were characterized using NMR spectroscopy as well as computational approaches8 with emphasis mainly on the large‐scale conformational exchange associated with the opening of the nucleotide binding lid, which was elegantly identified as the rate‐limiting step for the overall reaction 9…”