The Temperature Dependence of the Kinetic Isotope Effects of Dihydrofolate Reductase from Thermotoga maritima Is Influenced by Intersubunit Interactions

摘要

Dihydrofolate reductase from the hyperthermophile Thermotoga maritima (TmDHFR) is uniquenamong structurally characterized chromosomal DHFRs in that it forms a stable homodimer. Dimerizationnis believed to play a key role in the high thermal stability of TmDHFR, which is reflected in a meltingntemperature in excess of 85 u0001C. The dimer interface of TmDHFR is composed of a hydrophobic core withncharged residues around the periphery. In particular, Lys129 of each subunit forms three-membered saltnbridges with Glu136 and Glu138 of the other subunit. To probe the role of these salt bridges in thendimerization and thermal stability of TmDHFR, we generated a series of variants (TmDHFR-K129E,nTmDHFR-E136K, TmDHFR-E138K, and TmDHFR-E136K/E138K) in which these residues were ex-nchanged for residues whose side chains bear the opposite charge.Our results indicate that these salt bridges arenkey for the high thermal stability of TmDHFR but are not a requirement for dimerization. Although the ratenof dihydrofolate reduction by TmDHFR is not significantly affected by the loss of theK129-E136-E138 saltnbridges, changes to the temperature dependence of the kinetic isotope effect on hydride transfer are observed.nThese changes are in agreement with the proposal that DHFR catalysis may be affected by changes to thenconformational ensemble of the enzyme rather than only to the coupling of protein motions to the reactionncoordinate.