Literature summary for 6.3.4.14 extracted from

Chou, C.Y.; Yu, L.P.; Tong, L.
Crystal structure of biotin carboxylase in complex with substrates and implications for its catalytic mechanism (2009), J. Biol. Chem., 284, 11690-11697.

Crystallization (Commentary)

Crystallization (Comment)	Organism
wild-type Escherichia coli biotin carboxylase and mutant E296A in complex with its substrates biotin, bicarbonate, and Mg-ADP, at 2.0 A resolution. Residue Glu296 is the general base that extracts the proton from bicarbonate, and Arg338 is the residue that stabilizes the enolate biotin intermediate in the carboxylation reaction. The B domain of biotin carboxylase is positioned closer to the active site, leading to a 2-A shift in the bound position of the adenine nucleotide and bringing it near the bicarbonate for catalysis. One of the oxygenatoms of bicarbonate is located in the correct position to initiate the nucleophilic attack on ATP to form the carboxyphosphate intermediate. The phosphate group, derived from decomposition of carboxyphosphate, is the general base that extracts the proton on this N1 atom	Escherichia coli

Crystallization (Comment)

Organism

wild-type Escherichia coli biotin carboxylase and mutant E296A in complex with its substrates biotin, bicarbonate, and Mg-ADP, at 2.0 A resolution. Residue Glu296 is the general base that extracts the proton from bicarbonate, and Arg338 is the residue that stabilizes the enolate biotin intermediate in the carboxylation reaction. The B domain of biotin carboxylase is positioned closer to the active site, leading to a 2-A shift in the bound position of the adenine nucleotide and bringing it near the bicarbonate for catalysis. One of the oxygenatoms of bicarbonate is located in the correct position to initiate the nucleophilic attack on ATP to form the carboxyphosphate intermediate. The phosphate group, derived from decomposition of carboxyphosphate, is the general base that extracts the proton on this N1 atom

Escherichia coli

Protein Variants

Protein Variants	Comment	Organism
E296A	50fold decrease in catalytic efficiency, crystallization data	Escherichia coli
R338A	250fold decrease in catalytic efficiency	Escherichia coli

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
16.2	-	HCO3-	pH 8.5, wild-type	Escherichia coli
18.9	-	HCO3-	pH 8.5, mutant R338A	Escherichia coli
22.7	-	biotin	pH 7.4, mutant E296A	Escherichia coli
35.1	-	biotin	pH 7.4 wild-type	Escherichia coli
41.8	-	biotin	pH 7.4, mutant R338A	Escherichia coli
57.5	-	HCO3-	pH 8.5, mutant E296A	Escherichia coli

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	P24182	-	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
ATP + biotin + HCO3-	-	Escherichia coli	ADP + phosphate + carboxybiotin	-	?

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.0019	-	HCO3-	pH 8.5, mutant R338A	Escherichia coli
0.0025	-	biotin	pH 7.4, mutant R338A	Escherichia coli
0.028	-	biotin	pH 7.4, mutant E296A	Escherichia coli
0.034	-	HCO3-	pH 8.5, mutant E296A	Escherichia coli
0.44	-	HCO3-	pH 8.5, wild-type	Escherichia coli
0.58	-	biotin	pH 7.4 wild-type	Escherichia coli

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.00006	-	biotin	pH 7.4, mutant R338A	Escherichia coli
0.0001	-	HCO3-	pH 8.5, mutant R338A	Escherichia coli
0.000594	-	HCO3-	pH 8.5, mutant E296A	Escherichia coli
0.00123	-	biotin	pH 7.4, mutant E296A	Escherichia coli
0.0165	-	biotin	pH 7.4 wild-type	Escherichia coli
0.0272	-	HCO3-	pH 8.5, wild-type	Escherichia coli