Role of long-range protein dynamics in different thymidylate synthase catalyzed reactions.

Thelma Abeysinghe,Amnon Kohen

doi:10.3390/ijms16047304

Thelma Abeysinghe, Amnon Kohen

Open Access

https://doi.org/10.3390/ijms16047304

Copy DOI

Abstract

Recent studies of Escherichia coli thymidylate synthase (ecTSase) showed that a highly conserved residue, Y209, that is located 8 Å away from the reaction site, plays a key role in the protein’s dynamics. Those crystallographic studies indicated that Y209W mutant is a structurally identical but dynamically altered relative to the wild type (WT) enzyme, and that its turnover catalytic rate governed by a slow hydride-transfer has been affected. The most challenging test of an examination of a fast chemical conversion that precedes the rate-limiting step has been achieved here. The physical nature of both fast and slow C-H bond activations have been compared between the WT and mutant by means of observed and intrinsic kinetic isotope effects (KIEs) and their temperature dependence. The findings indicate that the proton abstraction step has not been altered as much as the hydride transfer step. Additionally, the comparison indicated that other kinetic steps in the TSase catalyzed reaction were substantially affected, including the order of the substrate binding. Enigmatically, although Y209 is H-bonded to 3'-OH of 2'-deoxyuridine-5'-monophosphate (dUMP), its altered dynamics is more pronounced on the binding of the remote cofactor, (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate), revealing the importance of long-range dynamics of the enzymatic complex and its catalytic function.

Highlights

The role of protein dynamics in enzyme catalysis is one of the open questions in enzymology today.In particular, the participation of residues distal to the active site in catalyzing bond-activation at the active site is a topic of interest as it combines several enigmas: the long range communication in the enzyme, its “holistic” nature, and the different catalytic strategies applied by the same active site to catalyze different chemical conversions
The value of V/KA is always dependent on the concentration of B, while the value V/KB only depends on the concentration of A in the random mechanism (Figure 4B) but not in the ordered mechanism (Figure 4A)
The role of long-range amino acid communications and enzyme dynamics across proteins and its catalytic function is of significant contemporary interest and controversy

Summary

Introduction

The role of protein dynamics in enzyme catalysis is one of the open questions in enzymology today. The mechanism of TSase (Scheme 1) involves several chemical conversions including two C–H bond activations: a rate limiting hydride transfer (step 5) and a much faster proton transfer (step 4) Both these H-transfers have been studied in the WT enzyme via examination of the temperature dependence of intrinsic kinetic isotope effects (KIEintS) [16]. The vertical dashed line represents the DAD under which the Zero Point Energy (ZPE) is greater than the barrier height At such distances, the process of a wave function spreading from reactant well to product well is no longer “tunneling”, but one can still use the particle’s transmission probability analogously to the tunneling probability at longer DADs. In the non-adiabatic approximation (Figure 3), the heavy atom motion toward the TRS can be calculated as two parabolas (Marcus-parabolas). Along with the X-ray crystallographic data, we could correlate the kinetic findings and distinguish the effect of distal mutations on protein motions at different timescales that impact two different H-transfer reactions as well as other catalytic steps

Binding Mechanism of Y209W Mutant

Temperature Dependence of Intrinsic KIEs in the Y209W Mutant

Experimental Section

Conclusions