Kinetic Barrier to Enzyme Inhibition Is Manipulated by Dynamical Local Interactions in E. coli DHFR

Abstract

Dihydrofolate reductase (DHFR) is an important drug targetanda highly studied model protein for understanding enzyme dynamics.DHFR's crucial role in folate synthesis renders it an idealcandidate to understand protein function and protein evolution mechanisms.In this study, to understand how a newly proposed DHFR inhibitor,4 & PRIME;-deoxy methyl trimethoprim (4 & PRIME;-DTMP), alters evolutionarytrajectories, we studied interactions that lead to its superior performanceover that of trimethoprim (TMP). To elucidate the inhibition mechanismof 4 & PRIME;-DTMP, we first confirmed, both computationally and experimentally,that the relative binding free energy cost for the mutation of TMPand 4 & PRIME;-DTMP is the same, pointing the origin of the characteristicdifferences to be kinetic rather than thermodynamic. We then employedan interaction-based analysis by focusing first on the active siteand then on the whole enzyme. We confirmed that the polar modificationin 4 & PRIME;-DTMP induces additional local interactions with the enzyme,particularly, the M20 loop. These changes are propagated to the wholeenzyme as shifts in the hydrogen bond networks. To shed light on theallosteric interactions, we support our analysis with network-basedcommunity analysis and show that segmentation of the loop domain of inhibitor-bound DHFR must be avoided by a successful inhibitor.