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Literature summary for 1.1.2.3 extracted from

  • Tsai, C.L.; Gokulan, K.; Sobrado, P.; Sacchettini, J.C.; Fitzpatrick, P.F.
    Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base (2007), Biochemistry, 46, 7844-7851.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
recombinant wild-type Fcb2 and its recombinant flavin domain are expressed in Escherichia coli Saccharomyces cerevisiae

Crystallization (Commentary)

Crystallization (Comment) Organism
crystal structure of mutant H723Q bound with pyruvate is determined at 2.8 A Saccharomyces cerevisiae

Protein Variants

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 [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

Organism UniProt Comment Textmining
Saccharomyces cerevisiae P00175
-
-

Storage Stability

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 and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
L-lactate + potassium ferricyanide
-
Saccharomyces cerevisiae pyruvate + potassium ferrocyanide + H+
-
?

Synonyms

Synonyms Comment Organism
flavocytochrome b2
-
Saccharomyces cerevisiae

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Saccharomyces cerevisiae

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Saccharomyces cerevisiae