title: Chemical Ligation and Isotope Labeling to Locate Dynamic Effects during Catalysis by Dihydrofolate Reductase.
abstract: Chemical ligation has been used to alter motions in specific regions of dihydrofolate reductase from E. coli and to investigate the effects of localized motional changes on enzyme catalysis. Two isotopic hybrids were prepared; one with the mobile N-terminal segment containing heavy isotopes ((2) H, (13) C, (15) N) and the remainder of the protein with natural isotopic abundance, and the other one with only the C-terminal segment isotopically labeled. Kinetic investigations indicated that isotopic substitution of the N-terminal segment affected only a physical step of catalysis, whereas the enzyme chemistry was affected by protein motions from the C-terminal segment. QM/MM studies support the idea that dynamic effects on catalysis mostly originate from the C-terminal segment. The use of isotope hybrids provides insights into the microscopic mechanism of dynamic coupling, which is difficult to obtain with other studies, and helps define the dynamic networks of intramolecular interactions central to enzyme catalysis.
authors.affiliation: School of Chemistry, Cardiff University, Park Place, Cardiff, CF10 3AT (UK).
authors.forename: Louis Y P
authors.initials: LY
authors.lastname: Luk
authors.affiliation: Departament de Química Física i Analítica, Universitat Jaume I, 12071 Castelló (Spain).
authors.forename: J Javier
authors.initials: JJ
authors.lastname: Ruiz-Pernía
authors.affiliation: School of Chemistry, Cardiff University, Park Place, Cardiff, CF10 3AT (UK).
authors.forename: Aduragbemi S
authors.initials: AS
authors.lastname: Adesina
authors.affiliation: School of Chemistry, Cardiff University, Park Place, Cardiff, CF10 3AT (UK).
authors.forename: E Joel
authors.initials: EJ
authors.lastname: Loveridge
authors.affiliation: Departament de Química Física, Universitat de València, 46100 Burjassot (Spain). ignacio.tunon@uv.es.
authors.forename: Iñaki
authors.initials: I
authors.lastname: Tuñón
authors.affiliation: Departament de Química Física i Analítica, Universitat Jaume I, 12071 Castelló (Spain). moliner@uji.es.
authors.forename: Vincent
authors.initials: V
authors.lastname: Moliner
authors.affiliation: School of Chemistry, Cardiff University, Park Place, Cardiff, CF10 3AT (UK). allemannrk@cardiff.ac.uk.
authors.forename: Rudolf K
authors.initials: RK
authors.lastname: Allemann
mesh_terms: D002384:Catalysis; D007553:Isotope Labeling / Q000379:methods*; D008026:Ligation; D008958:Models, Molecular; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry*
chemical_list: D013762:Tetrahydrofolate Dehydrogenase
keywords: chemical ligation; enzyme catalysis; isotope effects; microscopic mechanisms; protein dynamics