Catalytic-rate improvement of a thermostable malate dehydrogenase by a subtle alteration in cofactor binding.
The nucleotide-binding fold of many NAD(+)-dependent dehydrogenases contains a conserved acidic amino acid residue which hydrogen-bonds with the 2'- and 3'-hydroxy groups of the adenine-ribose of the cofactor. This residue is highly conserved as aspartate in malate dehydrogenases, except in the thermophilic enzyme from Thermus aquaticus B (TaqMDH), which has glutamic acid-41 in the equivalent position. The catalytic mechanism was dissected to investigate the functional significance of this difference in TaqMDH with respect to a mutant enzyme where glutamic acid-41 was replaced by aspartic acid. The mutant enzyme was found to retain a high degree of protein structural stability to both thermal and chemical denaturation. When compared with the wild-type enzyme the mutant had a higher Km and Kd for both reduced and oxidized cofactors (NADH and NAD+) and a 2-3-fold increase in steady-state kcat in both assay directions. The rate-determining step for the reduction of oxaloacetate by wild-type TaqMDH was shown to be the rate of NAD+ release, which was about 2.5-fold higher for the mutant enzyme. This correlates well with the 1.8-fold higher steady-state kcat of the mutant enzyme and represents an improvement in the steady-state kcat of a thermophilic enzyme at moderate temperature by a conservative amino acid substitution which increases the rate of product release.
Division of Biotechnology, Centre for Applied Microbiology and Research, Porton, Salisbury, Wilts., U.K.
R M
RM
Alldread
D M
DM
Halsall
A R
AR
Clarke
T K
TK
Sundaram
T
T
Atkinson
M D
MD
Scawen
D J
DJ
Nicholls
D001224:Aspartic Acid / Q000235:genetics; D001483:Base Sequence; D002384:Catalysis; D004795:Enzyme Stability; D018698:Glutamic Acid / Q000235:genetics; D006358:Hot Temperature; D008291:Malate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009243:NAD / Q000378:metabolism*; D010071:Oxaloacetates / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012997:Solvents; D013329:Structure-Activity Relationship; D013824:Thermus / Q000201:enzymology*; D014783:Viscosity
D010071:Oxaloacetates; D011994:Recombinant Proteins; D012997:Solvents; D009243:NAD; D001224:Aspartic Acid; D018698:Glutamic Acid; D008291:Malate Dehydrogenase