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Literature summary for 3.5.2.6 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)

Crystallization (Comment) Organism
hanging drop method plasmid pBG66

Protein Variants

Protein Variants Comment Organism
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
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
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]

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

Organism

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

Purification (Commentary)

Purification (Comment) Organism
-
plasmid pBG66

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
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
-
?
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
-
?
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
-
?
penicillin G + H2O
-
plasmid pBG66 (2R,4S)-2-[(R)-carboxy[(phenylacetyl)amino]methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
-
?

Synonyms

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

Turnover Number [1/s]

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

kcat/KM [mM/s]

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