Crystallization (Comment) | Organism |
---|---|
hanging drop method | plasmid pBG66 |
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 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 | UniProt | Comment | Textmining |
---|---|---|---|
plasmid pBG66 | - |
contains the wild-type blaTEM-1 gene, expressed in Escherichia coli | - |
Purification (Comment) | Organism |
---|---|
- |
plasmid pBG66 |
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 | Comment | Organism |
---|---|---|
TEM-1 beta-lactamase | class A beta-lactamase | plasmid pBG66 |
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 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 |