Cloned (Comment) | Organism |
---|---|
recombinant wild-type Fcb2 and its recombinant flavin domain are expressed in Escherichia coli | Saccharomyces cerevisiae |
Crystallization (Comment) | Organism |
---|---|
crystal structure of mutant H723Q bound with pyruvate is determined at 2.8 A | Saccharomyces cerevisiae |
Protein Variants | Comment | Organism |
---|---|---|
H373Q | His373 acts as an active site base during the oxidation of lactate to pyruvate. The decrease of 3500fold in the rate constant for reduction of the enzyme-bound FMN by lactate confirms this part of the reaction as that most affected by the mutation. Primary deuterium and solvent kinetic isotope affects for the mutant enzyme are significantly smaller than the wild-type values, establishing that bond cleavage steps are less rate-limiting in H373Q flavocytochrome b2 than in wild-type. Structure of the mutant enzyme with pyruvate bound, determined at 2.8 A, shows that the orientation of pyruvate in the active site is altered from that seen in the wild-type enzyme. Active site residues Arg289, Asp292, and Leu286 have altered positions in the mutant protein. The combination of an altered active site and the small kinetic isotope effects is consistent with the slowest step in turnover being a conformational change involving a conformation in which lactate is bound unproductively | Saccharomyces cerevisiae |
H373Q | kcat (1/sec) (substrate:lactate): 0.031 (intact protein), 0.057 (flavin domain only), Km (mM) (substrate: lactate): 0.65 (intact protein), 0.25 (flavin domain only) | Saccharomyces cerevisiae |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.16 | - |
L-lactate | wild-type, intact protein | Saccharomyces cerevisiae | |
0.25 | - |
L-lactate | mutant H373Q, using flavin domain only | Saccharomyces cerevisiae | |
0.36 | - |
L-lactate | wild-type, using flavin domain only | Saccharomyces cerevisiae | |
0.65 | - |
L-lactate | mutant H373Q, intact protein | Saccharomyces cerevisiae |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Saccharomyces cerevisiae | P00175 | - |
- |
Storage Stability | Organism |
---|---|
-70°C, purified enzymes are stored in 100 mM potassium phosphate, 1 mM EDTA, and 20 mM D,L-lactate at pH 7.5 | Saccharomyces cerevisiae |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-lactate + potassium ferricyanide | - |
Saccharomyces cerevisiae | pyruvate + potassium ferrocyanide + H+ | - |
? |
Synonyms | Comment | Organism |
---|---|---|
flavocytochrome b2 | - |
Saccharomyces cerevisiae |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Saccharomyces cerevisiae |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.031 | - |
L-lactate | mutant H373Q, intact protein | Saccharomyces cerevisiae | |
0.057 | - |
L-lactate | mutant H373Q, using flavin domain only | Saccharomyces cerevisiae | |
200 | - |
L-lactate | wild-type, using flavin domain only | Saccharomyces cerevisiae | |
372 | - |
L-lactate | wild-type, intact protein | Saccharomyces cerevisiae |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Saccharomyces cerevisiae |