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

Cathleen Zeymer,D Flemming Hansen,Sabine Zimmermann,Nicolas D Werbeck,Jochen Reinstein

doi:10.1002/anie.201606238

Abstract

States along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK were captured and changes in the conformational heterogeneity of conserved active site arginine side‐chains were quantified by NMR spin‐relaxation methods. In addition to apo and ligand‐bound UmpK, a transition state analog (TSA) complex was utilized to evaluate the extent to which active site conformational entropy contributes to the transition state free energy. The catalytically essential arginine side‐chain guanidino groups were found to be remarkably rigid in the TSA complex, indicating that the enzyme has evolved to restrict the conformational freedom along its reaction path over the energy landscape, which in turn allows the phosphoryl transfer to occur selectively by avoiding side reactions.

Highlights

States along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UMP/CMP kinase (UmpK) were captured and changes in the conformational heterogeneity of conserved active site arginine side-chains were quantified by NMR spin-relaxation methods
Experimental information on the entropic contribution to the transition state free energy originating from conformational heterogeneity is still strikingly lacking.[3]
Backbone dynamics of the Nucleoside monophosphate (NMP) kinase adenylate kinase were characterized using NMR spectroscopy as well as computational approaches[8] 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]

Summary

Introduction

States along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK were captured and changes in the conformational heterogeneity of conserved active site arginine side-chains were quantified by NMR spin-relaxation methods. We focus on the central phosphoryl transfer reaction step of NMP kinases, using the UMP/CMP kinase (UmpK) from Dictyostelium discoideum, and turn the attention directly to the conformational heterogeneity of the catalytic site and the functional groups that actively participate in stabilizing the transition state, namely the positively charged side-chains of the six conserved arginine residues[5] (Figure 1).

Results

Conclusion