The temperature dependence of the kinetic isotope effects of dihydrofolate reductase from Thermotoga maritima is influenced by intersubunit interactions.
Dihydrofolate reductase from the hyperthermophile Thermotoga maritima (TmDHFR) is unique among structurally characterized chromosomal DHFRs in that it forms a stable homodimer. Dimerization is believed to play a key role in the high thermal stability of TmDHFR, which is reflected in a melting temperature in excess of 85 degrees C. The dimer interface of TmDHFR is composed of a hydrophobic core with charged residues around the periphery. In particular, Lys129 of each subunit forms three-membered salt bridges with Glu136 and Glu138 of the other subunit. To probe the role of these salt bridges in the dimerization and thermal stability of TmDHFR, we generated a series of variants (TmDHFR-K129E, TmDHFR-E136K, TmDHFR-E138K, and TmDHFR-E136K/E138K) in which these residues were exchanged for residues whose side chains bear the opposite charge. Our results indicate that these salt bridges are key for the high thermal stability of TmDHFR but are not a requirement for dimerization. Although the rate of dihydrofolate reduction by TmDHFR is not significantly affected by the loss of the K129-E136-E138 salt bridges, changes to the temperature dependence of the kinetic isotope effect on hydride transfer are observed. These changes are in agreement with the proposal that DHFR catalysis may be affected by changes to the conformational ensemble of the enzyme rather than only to the coupling of protein motions to the reaction coordinate.
School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, United Kingdom.
E Joel
EJ
Loveridge
Rudolf K
RK
Allemann
D001483:Base Sequence; D017931:DNA Primers; D019281:Dimerization; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013696:Temperature; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020124:Thermotoga maritima / Q000201:enzymology*
D017931:DNA Primers; D013762:Tetrahydrofolate Dehydrogenase