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Literature summary for 3.5.2.6 extracted from

  • Drawz, S.M.; Bethel, C.R.; Hujer, K.M.; Hurless, K.N.; Distler, A.M.; Caselli, E.; Prati, F.; Bonomo, R.A.
    The role of a second-shell residue in modifying substrate and inhibitor interactions in the SHV beta-lactamase: a study of ambler position Asn276 (2009), Biochemistry, 48, 4557-4566.
    View publication on PubMedView publication on EuropePMC

Protein Variants

Protein Variants Comment Organism
additional information mutagenesis is performed to better understand the basis of the inhibitor-resistant phenotype in SHV, examination of the role of a second-shell residue, Asn276 synthetic construct
N276D reduced susceptibility to ampicillin/clavulanate while maintaining high-level resistance to ampicillin. Slightly diminished kcat/Km for all substrates tested. 5-fold increase in Ki for clavulanate and a 40% reduction in kinact/KI synthetic construct

Inhibitors

Inhibitors Comment Organism Structure
(6Z)-7-oxo-6-(4H-pyrazolo[1,5-c][1,3]thiazol-2-ylmethylidene)-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid
-
synthetic construct
clavulanate
-
synthetic construct
meropenem
-
synthetic construct

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.021
-
nitrocefin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct
0.028
-
nitrocefin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct
0.029
-
cephalothin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct
0.077
-
piperacillin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct
0.083
-
cephalothin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct
0.105
-
piperacillin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct
0.134
-
ampicillin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct
0.183
-
ampicillin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct

Organism

Organism UniProt Comment Textmining
synthetic construct
-
using the template blaSHV-1 gene in the phagemid vector pBC SK(-), degeneration of oligonucleotides at Ambler position 276 to make the full complement of amino acid substitutions. After PCR mutagenesis, electroporation of the resultant plasmids into Escherichia coli
-

Purification (Commentary)

Purification (Comment) Organism
-
synthetic construct

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ampicillin + H2O
-
synthetic construct (2R,4S)-2-[(R)-[[(2R)-2-amino-2-phenylacetyl]amino](carboxy)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
-
?
cephalothin + H2O
-
synthetic construct (2R)-5-[(acetyloxy)methyl]-2-[(R)-carboxy[(thiophen-2-ylacetyl)amino]methyl]-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid
-
?
nitrocefin + H2O
-
synthetic construct (2R)-2-[(R)-carboxy[(thiophen-2-ylacetyl)amino]methyl]-5-[(E)-2-(2,4-dinitrophenyl)ethenyl]-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid
-
?
piperacillin + H2O
-
synthetic construct (2R,4S)-2-[(R)-carboxy[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]-2-phenylacetyl]amino]methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
-
?

Synonyms

Synonyms Comment Organism
blaSHV-1
-
synthetic construct
SHV-1
-
synthetic construct

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
10
-
cephalothin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct
14
-
cephalothin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct
137
-
nitrocefin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct
237
-
nitrocefin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct
898
-
piperacillin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct
976
-
piperacillin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct
989
-
ampicillin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct
3100
-
ampicillin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
120
-
cephalothin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct
470
-
cephalothin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct
5000
-
nitrocefin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct
7000
-
ampicillin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct
9000
-
piperacillin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct
11000
-
nitrocefin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct
12000
-
piperacillin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct
17000
-
ampicillin pH 7.4, 22°C, wild-type enzyme SHV-1 synthetic construct