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 (2H, 13C, 15N) 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.

Protein Mass Effects on Formate Dehydrogenase

“…33 However, these studies show different effects of isotopic substitution depending on the enzymatic system. Beyond assessing the importance of protein dynamics in enzyme catalysis 34-36 heavy enzyme studies have also been used in the development of inhibitors for pharmaceutical applications as well. 37,38 Nevertheless, further investigation is vital to obtain a comprehensive understanding of the correlation between altered fast protein dynamics and the catalyzed reaction along with other effects of isotopic substitution on enzyme catalysis.…”

Aligator: A computational tool for optimizing total chemical synthesis of large proteins

“…Some recent examples include ubiquitin 36 , alpha-synuclein 37 , histones 38 , and membrane proteins 39 . Additional examples include fundamental ubiquitin biology 40 , proteins with selective isotopic labeling 41 , site-specific installation of fluors (e.g., FRET pairs) 42 , and interesting scaffold approaches 43 .…”

Pinpointing Dynamic Coupling in Enzymes for Efficient Drug Design

“…These apparently contradictory results demonstrate the delicate nature of motional coupling to catalysis. We have refined this global picture of motional coupling by isotopically labeling specific segments of an enzyme and therefore experimentally determining which parts of the enzyme show motional coupling [19]. Two isotopic hybrids of DHFR were prepared by chemical ligation techniques [20], one in which the mobile N-terminal segment contained heavy isotopes while the remainder of the protein was of natural isotopic abundance, and one in which only the C-terminal region was isotopically labeled.…”

“…Two isotopic hybrids of DHFR were prepared by chemical ligation techniques [20], one in which the mobile N-terminal segment contained heavy isotopes while the remainder of the protein was of natural isotopic abundance, and one in which only the C-terminal region was isotopically labeled. These experiments revealed that the N-terminal region is only involved in beneficial millisecond conformational motions, while in the C-terminal region fast dynamic motions couple unfavorably to barrier crossing, thereby slowing the reaction [19]. The combination of chemical ligation and heavy enzyme approaches has shown for the first time that different parts of an enzyme affect different aspects of its function.…”