Advanced Spectroscopy and APBS Modeling for Determination of the Role of His190 and Trp103 in Mouse Thymidylate Synthase Interaction with Selected dUMP Analogues.

Małgorzata Prokopowicz,Zbigniew Zieliński,Fiona Gordon,Alan Ryder,Justyna Sobich,Piotr Maj,Adam Jarmuła,Yannick Casamayou-Boucau,Joanna Cieśla,Piotr Fita,Wojciech Rode

doi:10.3390/ijms22052661

Abstract

A homo-dimeric enzyme, thymidylate synthase (TS), has been a long-standing molecular target in chemotherapy. To further elucidate properties and interactions with ligands of wild-type mouse thymidylate synthase (mTS) and its two single mutants, H190A and W103G, spectroscopic and theoretical investigations have been employed. In these mutants, histidine at position 190 and tryptophan at position 103 are substituted with alanine and glycine, respectively. Several emission-based spectroscopy methods used in the paper demonstrate an especially important role for Trp 103 in TS ligands binding. In addition, the Advanced Poisson–Boltzmann Solver (APBS) results show considerable differences in the distribution of electrostatic potential around Trp 103, as compared to distributions observed for all remaining Trp residues in the mTS family of structures. Together, spectroscopic and APBS results reveal a possible interplay between Trp 103 and His190, which contributes to a reduction in enzymatic activity in the case of H190A mutation. Comparison of electrostatic potential for mTS complexes, and their mutants, with the substrate, dUMP, and inhibitors, FdUMP and N-OH-dCMP, suggests its weaker influence on the enzyme–ligand interactions in NOH-dCMP-mTS compared to dUMP-mTS and FdUMP-mTS complexes. This difference may be crucial for the explanation of the ”abortive reaction” inhibitory mechanism of NOH-dCMP towards TS. In addition, based on structural analyses and the H190A mutant capacity to form a denaturation-resistant complex with N-OH-dCMP in the mTHF-dependent reaction, His190 is apparently responsible for a strong preference of the enzyme active center for the anti rotamer of the imino inhibitor form.

Highlights

Introduction conditions of the Creative CommonsThymidylate synthase (TS) is an enzyme with a long-standing history of a dedicated comprehensive research
It is worth mentioning that in spite of the differences seen in their fluorescence spectra, the WT mouse thymidylate synthase (mTS) enzyme and its mutants have the absorption maxima located at the same wavelength, λ = 282 nm
The non-negligible Trp absorption and the relatively long excited-state lifetime of other Trps in 290–315 nm range are long enough for homoFRET to occur in mTS/H190A

Summary

Introduction

Introduction conditions of the Creative CommonsThymidylate synthase (TS) is an enzyme with a long-standing history of a dedicated comprehensive research.

Objectives

Results

Discussion

Conclusion