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Literature summary for 5.1.1.3 extracted from

  • Mehboob, S.; Guo, L.; Fu, W.; Mittal, A.; Yau, T.; Truong, K.; Johlfs, M.; Long, F.; Fung, L.; Johnson, M.
    Glutamate racemase dimerization inhibits dynamic conformational flexibility and reduces catalytic rates (2009), Biochemistry, 48, 7045-7055.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 5.1.1.3

Application

Application Comment Organism
drug development einzyme is potentially an attractive target for the development of new antibacterial agents because of being an essential enzyme Bacillus anthracis

Cloned(Commentary)

Cloned (Comment) Organism
recombinant wild type and mutants are expressed in BL21(DE3) Escherichia coli cells Bacillus anthracis

Protein Variants

Protein Variants Comment Organism
K106A production by site-directed mutagenesis, catalytic rate is higher than that of the wild type in D-glutamine to L-glutamine direction, kinetics in the L-glutamine to D-glutamine direction is not as significantly affected with an a 2-3fold increase in the overall catalytic efficiency, disruption of the dimer interface Bacillus anthracis
K29A production by site-directed mutagenesis, catalytic rate is higher than that of the wild type in D-glutamine to L-glutamine direction, kinetics in the L-glutamine to D-glutamine direction is not as significantly affected with a 2-3fold increase in the overall catalytic efficiency, disruption of the dimer interface Bacillus anthracis
P99A production by site-directed mutagenesis, catalytic rate is higher than that of the wild type in D-glutamine to L-glutamine direction, kinetics in the L-glutamine to D-glutamine direction is not as significantly affected with a 2-3fold increase in the overall catalytic efficiency, disruption of the dimer interface Bacillus anthracis
Q86A production by site-directed mutagenesis, catalytic rate is higher than that of the wild type in D-glutamine to L-glutamine direction, kinetics in the L-glutamine to D-glutamine direction is not as significantly affected with an at most 2-3fold increase in the overall catalytic efficiency, disruption of the dimer interface Bacillus anthracis
R214A production by site-directed mutagenesis, catalytic rate is higher than that of the wild type in D-glutamine to L-glutamine direction, kinetics in the L-glutamine to D-glutamine direction is not as significantly affected with a 2-3fold increase in the overall catalytic efficiency, disruption of the dimer interface Bacillus anthracis
R214A/K106A production by site-directed mutagenesis, catalytic rate is higher than that of the wild type in D-glutamine to L-glutamine direction, kinetics in the L-glutamine to D-glutamine direction is not as significantly affected with a 2-3fold increase in the overall catalytic efficiency, disruption of the dimer interface Bacillus anthracis
R25A production by site-directed mutagenesis, catalytic rate is higher than that of the wild type in D-glutamine to L-glutamine direction, kinetics in the L-glutamine to D-glutamine direction is not as significantly affected with a 2-3fold increase in the overall catalytic efficiency, disruption of the dimer interface Bacillus anthracis
Y221A production by site-directed mutagenesis, catalytic rate is higher than that of the wild type in D-glutamine to L-glutamine direction, kinetics in the L-glutamine to D-glutamine direction is not as significantly affected with an at most 2-3fold increase in the overall catalytic efficiency, disruption of the dimer interface Bacillus anthracis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.07
-
 D-glutamate recombinant wild type enzyme, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/mL diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
0.1
-
 D-glutamate mutant Q86A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
0.13
-
 D-glutamate mutant Y221A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
0.14
-
 D-glutamate mutant R25A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
0.16
-
 D-glutamate mutant R214A/K106A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
0.17
-
 D-glutamate mutant P99A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
0.25
-
 D-glutamate mutant K29A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
0.3
-
 D-glutamate mutant R214A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
0.42
-
 D-glutamate mutant K106A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
4.1
-
 L-glutamate mutant P99A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
5.8
-
 L-glutamate mutant R25A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
6.1
-
 L-glutamate recombinant RacE2 wild type enzyme, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
7.2
-
 L-glutamate mutant K29A, in 10 mM potassium phosphate (pH 8.2)and 0.2 mM dithiothreitol Bacillus anthracis
8.8
-
 L-glutamate mutant Q86A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
9.9
-
 L-glutamate mutant Y221A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
10.1
-
 L-glutamate mutant R214A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
10.8
-
 L-glutamate mutant K106A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
11
-
 L-glutamate mutant R214A/K106A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-glutamate Bacillus anthracis
-
 D-glutamate
-
 r

Organism

Organism UniProt Comment Textmining
Bacillus anthracis
-
-
-

Purification (Commentary)

Purification (Comment) Organism
by using nickel-nitrilotriacetic acid affinity chromatography and gel filtration Bacillus anthracis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
L-glutamate
-
 Bacillus anthracis D-glutamate
-
 r

Subunits

Subunits Comment Organism
dimer determined by gel filtration, RacE2 exist as a dimer in solution, but the monomeric enzyme is more active than the dimeric form Bacillus anthracis
monomer mutants R25A and K106A/R214A are completely monomeric at the concentration of 5 mg/ml Bacillus anthracis

Synonyms

Synonyms Comment Organism
glutamate racemase
-
 Bacillus anthracis
RACE
-
 Bacillus anthracis
RacE2
-
 Bacillus anthracis

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
49
-
 Tm value (midpoint of thermal unfolding) of the wild type enzyme Bacillus anthracis
51
-
 Tm value (midpoint of thermal unfolding) of the R25A mutant Bacillus anthracis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
780
-
 D-glutamate recombinant wild type enzyme, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
2160
-
 D-glutamate mutant Q86A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
2760
-
 D-glutamate mutant Y221A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
5220
-
 D-glutamate mutant K106A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
6660
-
 D-glutamate mutant R214A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
7440
-
 D-glutamate mutant K29A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
7560
-
 D-glutamate mutant R25A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
7680
-
 D-glutamate mutant P99A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
7980
-
 D-glutamate mutant R214A/K106A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
116600
-
 L-glutamate recombinant wild type enzyme, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
144000
-
 L-glutamate mutant Q86A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
164400
-
 L-glutamate mutant P99A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
219600
-
 L-glutamate mutant K106A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
252000
-
 L-glutamate mutant Y221A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
278400
-
 L-glutamate mutant R214A/K106A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
294000
-
 L-glutamate mutant R25A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
306000
-
 L-glutamate mutant K29A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
355200
-
 L-glutamate mutant R214A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis

General Information

General Information Comment Organism
physiological function peptidoglycan synthesis Bacillus anthracis

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.0031
-
 D-glutamate recombinant wild type enzyme, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
0.0035
-
 D-glutamate mutant K106A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
0.0045
-
 L-glutamate mutant Q86A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
0.0053
-
 L-glutamate recombinant wild type enzyme, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
0.0056
-
 L-glutamate mutant K106A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
0.0059
-
 D-glutamate mutant Y221A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
0.006
-
 D-glutamate mutant Q86A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
0.0062
-
 D-glutamate mutant R214A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
0.007
-
 L-glutamate mutant R214A/K106A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
0.0071
-
 L-glutamate mutant Y221A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
0.0083
-
 D-glutamate mutant K29A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
0.0098
-
 L-glutamate mutant R214A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
0.0111
-
 L-glutamate mutant P99A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
0.0118
-
 L-glutamate mutant K29A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
0.0125
-
 D-glutamate mutant P99A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
0.0139
-
 D-glutamate mutant R214A/K106A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis
0.014
-
 L-glutamate mutant R25A, in 10 mM potassium phosphate (pH 8.2) and 0.2 mM dithiothreitol Bacillus anthracis
0.015
-
 D-glutamate mutant R25A, reactions are conducted at 25°C in the assay mixture that contains 5 mM NAD+, 2.5 mM ADP, 50 mM CHES buffer (pH 9.2), 0.65 mM iodonitrotetrazolium chloride, 37.5 units/ml L-glutamate dehydrogenase, 2 units/ml diaphorase, and 0.05 mM D-glutamate Bacillus anthracis