title: Effects of pressure on enzyme function of Escherichia coli dihydrofolate reductase.
abstract: To elucidate the effects of pressure on the function of Escherichia coli dihydrofolate reductase (DHFR), the enzyme activity and the dissociation constants of substrates and cofactors were measured at pressures up to 250 MPa at 25 degrees C and pH 7.0. The enzyme activity decreased with increasing pressure, accompanying the activation volume of 7.8 ml mol(-1). The values of the Michaelis constant (K(m)) for dihydrofolate and NADPH were slightly higher at 200 MPa than at atmospheric pressure. The hydride-transfer step was insensitive to pressure, as monitored by the effects of the deuterium isotope of NADPH on the reaction velocity. The dissociation constants of substrates and cofactors increased with pressure, producing volume reductions from 6.5 ml mol(-1) (tetrahydrofolate) to 33.5 ml mol(-1) (NADPH). However, the changes in Gibbs free energy with dissociation of many ligands showed different pressure dependences below and above 50 MPa, suggesting conformational changes of the enzyme at high pressure. The enzyme function at high pressure is discussed based on the volume levels of the intermediates and the candidates for the rate-limiting process.
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Eiji
authors.initials: E
authors.lastname: Ohmae
authors.forename: Mineyuki
authors.initials: M
authors.lastname: Tatsuta
authors.forename: Fumiyoshi
authors.initials: F
authors.lastname: Abe
authors.forename: Chiaki
authors.initials: C
authors.lastname: Kato
authors.forename: Naoki
authors.initials: N
authors.lastname: Tanaka
authors.forename: Shigeru
authors.initials: S
authors.lastname: Kunugi
authors.forename: Kunihiko
authors.initials: K
authors.lastname: Gekko
mesh_terms: D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D011312:Pressure; D011487:Protein Conformation; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D008024:Ligands; D013762:Tetrahydrofolate Dehydrogenase