Role of threonine 190 in modulating the catalytic function of malate dehydrogenase from a thermophile Thermus flavus.
Random mutagenesis of malate dehydrogenase from a thermophilic bacterium, Thermus flavus AT-62, had revealed that a Thr190----Ile replacement near the essential catalytic residue His187 caused marked modulation of the catalytic properties. For further exploration of a role of the residue at this position, this residue was substituted with each of the other amino acids by site-directed mutagenesis. Most of the mutations except for substitution with Ser caused increases in Km for oxaloacetate and increases Ki for oxaloacetate of 2-110 times. Substitution with His or Pro was characterized by the complete loss of substrate inhibition, along with a marked increase in Km for oxaloacetate. Kinetic analyses of the native and altered malate dehydrogenases at various pHs revealed that both Km and Ki for oxaloacetate decreased proportionally to the decrease in pH from 8.40 to 5.75, whereas kcat was nearly constant within the pH range. Apparent shifts of the optimum pH values toward acidity observed with most of the altered malate dehydrogenases were attributed to the increase in Ki, which facilitated the release from the substrate inhibition at a lower pH. Replacement of Thr310, a possible counterpart with which Thr190 forms a hydrogen bond, by Ile caused changes in the catalytic properties similar to those of the Thr190-substituted enzymes. These results suggest that not only the loss of the hydrogen bond between Thr190 and Thr310 but also properties of the residues introduced at position 190 cause modulation of the catalytic properties, probably through dislocation of the loop structure that contains the catalytic residue His187.
Nishiyama
M
M
Department of Agricultural Chemistry, University of Tokyo, Japan.
Shimada
K
K
Horinouchi
S
S
Beppu
T
T
D001483:Base Sequence; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010071:Oxaloacetates / Q000378:metabolism; D010957:Plasmids; D013824:Thermus / Q000201:enzymology*; D013912:Threonine / Q000378:metabolism*
D010071:Oxaloacetates; D013912:Threonine; D008291:Malate Dehydrogenase