title: Hydride Transfer in DHFR by Transition Path Sampling, Kinetic Isotope Effects, and Heavy Enzyme Studies.
abstract: Escherichia coli dihydrofolate reductase (ecDHFR) is used to study fundamental principles of enzyme catalysis. It remains controversial whether fast protein motions are coupled to the hydride transfer catalyzed by ecDHFR. Previous studies with heavy ecDHFR proteins labeled with (13)C, (15)N, and nonexchangeable (2)H reported enzyme mass-dependent hydride transfer kinetics for ecDHFR. Here, we report refined experimental and computational studies to establish that hydride transfer is independent of protein mass. Instead, we found the rate constant for substrate dissociation to be faster for heavy DHFR. Previously reported kinetic differences between light and heavy DHFRs likely arise from kinetic steps other than the chemical step. This study confirms that fast (femtosecond to picosecond) protein motions in ecDHFR are not coupled to hydride transfer and provides an integrative computational and experimental approach to resolve fast dynamics coupled to chemical steps in enzyme catalysis.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona , P.O. Box 210041, 1306 East University Boulevard, Tucson, Arizona 85721, United States.
authors.forename: Dimitri
authors.initials: D
authors.lastname: Antoniou
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona , P.O. Box 210041, 1306 East University Boulevard, Tucson, Arizona 85721, United States.
authors.forename: Steven D
authors.initials: SD
authors.lastname: Schwartz
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: Vern L
authors.initials: VL
authors.lastname: Schramm
mesh_terms: D002247:Carbon Isotopes / Q000032:analysis / Q000378:metabolism; D003903:Deuterium / Q000032:analysis / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009587:Nitrogen Isotopes / Q000032:analysis / Q000378:metabolism; D011487:Protein Conformation; D013696:Temperature; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D003903:Deuterium; D013762:Tetrahydrofolate Dehydrogenase