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Literature summary extracted from

  • Brown, N.G.; Shanker, S.; Prasad, B.V.; Palzkill, T.
    Structural and biochemical evidence that a TEM-1 beta-lactamase N170G active site mutant acts via substrate-assisted catalysis (2009), J. Biol. Chem., 284, 33703-33712.
    View publication on PubMedView publication on EuropePMC

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.5.2.6 hanging drop method plasmid pBG66

Protein Variants

EC Number Protein Variants Comment Organism
3.5.2.6 N166A/170G kcat/KM for ampicillin is 725fold lower than wild-type value, kcat/KM for penicillin G is 850fold lower than wild-type value plasmid pBG66
3.5.2.6 N170A kcat/KM for ampicillin is 17fold lower than wild-type value, kcat/KM for penicillin G is 6fold lower than wild-type value, kcat/KM for cephalexin is 10fold lower than wild-type value, kcat/KM for cephalothin is 5fold lower than wild-type value plasmid pBG66
3.5.2.6 N170G very efficient at hydrolyzing substrates that contain a primary amine in the antibiotic R-group that would be close to the Asn170 side chain in the acyl-intermediate. The X-ray structure of the N170G enzyme indicates that the position of an active site water important for deacylation is altered compared with the wild-type enzyme. N170G TEM-1 hydrolyzes ampicillin efficiently because of substrate-assisted catalysis where the primary amine of the ampicillin R-group positions the hydrolytic water and allows for efficient deacylation. kcat/KM for ampicillin is 3.5fold lower than wild-type value, kcat/KM for penicillin G is 1.6fold lower than wild-type value, kcat/KM for cephalexin is 1.7fold lower than wild-type value, kcat/KM for cephalothin is 40fold lower than wild-type value plasmid pBG66

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.5.2.6 0.003
-
 penicillin G pH and temperature not specified in the publication, mutant enzyme N170G plasmid pBG66
3.5.2.6 0.008
-
 penicillin G pH and temperature not specified in the publication, mutant enzyme N170A plasmid pBG66
3.5.2.6 0.02
-
 penicillin G pH and temperature not specified in the publication, mutant enzyme N166A/N170G plasmid pBG66
3.5.2.6 0.024
-
 ampicillin pH and temperature not specified in the publication, mutant enzyme N166A/N170G plasmid pBG66
3.5.2.6 0.028
-
 penicillin G pH and temperature not specified in the publication, wild-type enzyme plasmid pBG66
3.5.2.6 0.038
-
 ampicillin pH and temperature not specified in the publication, wild-type enzyme plasmid pBG66
3.5.2.6 0.056
-
 ampicillin pH and temperature not specified in the publication, mutant enzyme N170G plasmid pBG66
3.5.2.6 0.13
-
 ampicillin pH and temperature not specified in the publication, mutant enzyme N170A plasmid pBG66
3.5.2.6 0.182
-
 cephalothin pH and temperature not specified in the publication, wild-type enzyme plasmid pBG66
3.5.2.6 1.162
-
 cephalexin pH and temperature not specified in the publication, wild-type enzyme plasmid pBG66
3.5.2.6 1.5
-
 cephalothin pH and temperature not specified in the publication, mutant enzyme N170G plasmid pBG66
3.5.2.6 1.56
-
 cephalexin pH and temperature not specified in the publication, mutant enzyme N170A plasmid pBG66
3.5.2.6 2.59
-
 cephalexin pH and temperature not specified in the publication, mutant enzyme N170G plasmid pBG66

Organism

EC Number Organism UniProt Comment Textmining
3.5.2.6 plasmid pBG66
-
 contains the wild-type blaTEM-1 gene, expressed in Escherichia coli
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.5.2.6
-
 plasmid pBG66

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.5.2.6 ampicillin + H2O
-
 plasmid pBG66 (2R,4S)-2-[(R)-[[(2R)-2-amino-2-phenylacetyl]amino](carboxy)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
-
 ?
3.5.2.6 cephalexin + H2O
-
 plasmid pBG66 (2R)-2-[(R)-[[(2R)-2-amino-2-phenylacetyl]amino](carboxy)methyl]-5-methyl-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid
-
 ?
3.5.2.6 cephalothin + H2O
-
 plasmid pBG66 (2R)-5-[(acetyloxy)methyl]-2-[(R)-carboxy[(thiophen-2-ylacetyl)amino]methyl]-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid
-
 ?
3.5.2.6 penicillin G + H2O
-
 plasmid pBG66 (2R,4S)-2-[(R)-carboxy[(phenylacetyl)amino]methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
-
 ?

Synonyms

EC Number Synonyms Comment Organism
3.5.2.6 TEM-1 beta-lactamase class A beta-lactamase plasmid pBG66

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.5.2.6 0.8
-
 penicillin G pH and temperature not specified in the publication, mutant enzyme N166A/N170G plasmid pBG66
3.5.2.6 0.9
-
 ampicillin pH and temperature not specified in the publication, mutant enzyme N166A/N170G plasmid pBG66
3.5.2.6 1
-
 cephalexin pH and temperature not specified in the publication, mutant enzyme N170A plasmid pBG66
3.5.2.6 13
-
 cephalexin pH and temperature not specified in the publication, wild-type enzyme plasmid pBG66
3.5.2.6 16
-
 cephalexin pH and temperature not specified in the publication, mutant enzyme N170G plasmid pBG66
3.5.2.6 22
-
 ampicillin pH and temperature not specified in the publication, mutant enzyme N170A plasmid pBG66
3.5.2.6 31
-
 cephalothin pH and temperature not specified in the publication, mutant enzyme N170G plasmid pBG66
3.5.2.6 45
-
 penicillin G pH and temperature not specified in the publication, mutant enzyme N170A plasmid pBG66
3.5.2.6 64
-
 penicillin G pH and temperature not specified in the publication, mutant enzyme N170G plasmid pBG66
3.5.2.6 146
-
 cephalothin pH and temperature not specified in the publication, wild-type enzyme plasmid pBG66
3.5.2.6 467
-
 ampicillin pH and temperature not specified in the publication, mutant enzyme N170G plasmid pBG66
3.5.2.6 950
-
 penicillin G pH and temperature not specified in the publication, wild-type enzyme plasmid pBG66
3.5.2.6 1085
-
 ampicillin pH and temperature not specified in the publication, wild-type enzyme plasmid pBG66

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.5.2.6 10
-
 cephalexin pH and temperature not specified in the publication, wild-type enzyme plasmid pBG66
3.5.2.6 800
-
 cephalothin pH and temperature not specified in the publication, wild-type enzyme plasmid pBG66
3.5.2.6 29000
-
 ampicillin pH and temperature not specified in the publication, wild-type enzyme plasmid pBG66
3.5.2.6 34000
-
 penicillin G pH and temperature not specified in the publication, wild-type enzyme plasmid pBG66