Information on EC 1.1.1.363 - glucose-6-phosphate dehydrogenase [NAD(P)+]

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The expected taxonomic range for this enzyme is: Leuconostoc mesenteroides

EC NUMBER
COMMENTARY hide
1.1.1.363
-
RECOMMENDED NAME
GeneOntology No.
glucose-6-phosphate dehydrogenase [NAD(P)+]
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
D-glucose 6-phosphate + NAD(P)+ = 6-phospho-D-glucono-1,5-lactone + NAD(P)H + H+
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Pentose phosphate pathway
-
-
Metabolic pathways
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-
Biosynthesis of secondary metabolites
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Biosynthesis of antibiotics
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SYSTEMATIC NAME
IUBMB Comments
D-glucose-6-phosphate:NAD(P)+ 1-oxidoreductase
The enzyme catalyses a step of the pentose phosphate pathway. The enzyme from the Gram-positive bacterium Leuconostoc mesenteroides prefers NADP+ while the enzyme from the Gram-negative bacterium Gluconacetobacter xylinus prefers NAD+. cf. EC 1.1.1.49, glucose-6-phosphate dehydrogenase (NADP+) and EC 1.1.1.388, glucose-6-phosphate dehydrogenase (NAD+).
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-deoxy-D-glucose 6-phosphate + NAD+
?
show the reaction diagram
-
low activity
-
-
?
D-galactose 6-phosphate + NAD+
?
show the reaction diagram
-
low activity
-
-
?
D-glucose 6-phosphate + NAD(P)+
6-phospho-D-glucono-1,5-lactone + NAD(P)H + H+
show the reaction diagram
-
-
-
-
?
D-glucose 6-phosphate + NAD+
6-phospho-D-glucono-1,5-lactone + NADH + H+
show the reaction diagram
D-glucose 6-phosphate + NADP+
6-phospho-D-glucono-1,5-lactone + NADPH + H+
show the reaction diagram
D-glucose 6-phosphate + NADP+ 2',3'-dialdehyde
6-phospho-D-glucono-1,5-lactone + NADPH 2',3'-dialdehyde + H+
show the reaction diagram
-
-
-
-
?
D-glucose 6-phosphate + thionicotinamide-NAD+
6-phospho-D-glucono-1,5-lactone + thionicotinamide-NADH + H+
show the reaction diagram
-
-
-
-
?
D-glucose 6-phosphate + thionicotinamide-NADP+
6-phospho-D-glucono-1,5-lactone + thionicotinamide-NADPH + H+
show the reaction diagram
-
-
-
-
?
D-glucose 6-sulfate + NAD+
?
show the reaction diagram
-
low activity
-
-
?
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
D-glucose 6-phosphate + NAD+
6-phospho-D-glucono-1,5-lactone + NADH + H+
show the reaction diagram
-
-
-
-
?
D-glucose 6-phosphate + NADP+
6-phospho-D-glucono-1,5-lactone + NADPH + H+
show the reaction diagram
-
-
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADP+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2',5'-ADP
-
NADP+-competitive and NAD+-noncompetitive inhibition
4-hydroxy-2-nonenal
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pseudo first-order loss of enzyme activity. The pH dependence of the inactivation rate exhibits an inflection around pH 10, and the enzyme is protected from inactivation by glucose 6-phosphate. Loss of enzyme activity corresponds with the formation of one carbonyl function per enzyme subunit and the appearance of a lysine-4-hydroxy-2-nonenal adduct
acetyl-CoA
citrate
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incubation of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with Fe2+ and citrate results in rapid O2-dependent inactivation of the enzyme. The Fe(2+)-citrate complex binds to the glucose 6-phosphate binding site and then undergoes reaction with H2O2 formed in solution leading to the oxidative modification of amino acids essential for enzyme activity
CoA
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3.4 mM, 12% inhibition of NADP+-dependent reaction, 82% inhibition of NAD+-dependent reaction
D-glucose 1-phosphate
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a substrate-competitive inhibitor, that lowers the dissociation constant and maximum fluorescence quenching for NAD+ but not for NADP+
D-glucose 6-phosphate
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high concentrations inhibit the NADP+-linked reaction in the dual wavelength assay (a method employing a mixture of one coenzyme and the thionicotinamide analog of the other coenzyme). Such inhibition is not observed in conventional assays using either NADP+ or thionicotinamide-NADP+
H2O2
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; the enzyme is rapidly inactivated by micromolar concentrations of Fe2+ and H2O2. Inactivation correlates with the formation of one carbonyl functionality/enzyme subunit, indicating that inactivation is the result of site-specific oxidative modification. Fe2+ binds to the glucose 6-phosphate binding site and interaction of the enzyme-bound Fe2+ with H2O2 leads to the oxidative modification of amino acids essential for enzyme activity. Partially inactivated enzyme remains predominantly in the dimeric form, and no change in the apparent affinity of the remaining active subunits for substrate is observed. Partial inactivation leads to a decrease in the thermal stability of the remaining activity. This decrease in thermal stability could be largely overcome by the addition of glucose 6-phosphate. Thus, although exposure to H2O2 and Fe2+ results in the irreversible inactivation of the enzyme, the resulting modification is selective, leads to the formation of heterodimers of both active and inactive subunits, and does not appear to cause large scale structural changes
malonyl-CoA
-
2.4 mM, no inhibition of NADP+-dependent reaction, 14% inhibition of NAD+-dependent reaction
N'-methylnicotinamide
-
-
NADP+
NADP+ 2',3'-dialdehyde
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irreversible inactivation in absence of substrate. The inactivation is first order with respect to NADP+ concentration and follows saturation kinetics, indicating that the enzyme initially forms a reversible complex with the inhibitor followed by covalent modification. NADP+ and NAD+ protect the enzyme from inactivation. One molecule of NADP+ 2',3'-dialdehyde binds per subunit of glucose-6-phosphate dehydrogenase when the enzyme is completely inactivated
NADPH
oleyl-CoA
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2.0 mM, 9% inhibition of NADP+-dependent reaction, 90% inhibition of NAD+-dependent reaction
palmitoyl-CoA
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inhibition is greatly diminished at high glucose 6-phosphate concentration
pyridoxal 5'-diphospho-5'-adenosine
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inhibits competitively with respect to glucose 6-phosphate and noncompetitively with respect to NAD+ or NADP+. 0.85 mol of pyridoxal 5'-diphospho-5'-adenosine is required for complete inactivation. Lys21 and Lys343 are the sites of pyridoxal 5'-diphospho-5'-adenosine interaction. Both glucose 6-phosphate and NAD+ protect both lysyl residues against this covalent modification
pyridoxal 5'-phosphate
vanadate
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inhibition by vanadate dimer and tetramer. The inhibition by vanadate is competitive with respect to NAD+ or NADP+ and noncompetitive (a mixed type) with respect to glucose 6-phosphate when NAD+ or NADP+ are cofactors. The vanadate dimer is the major inhibiting species with respect to NADP+. The vanadate tetramer is the major inhibiting species with respect to glucose 6-phosphate and with respect to NAD+. No inhibition by monomeric vanadate
additional information
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no inhibition by iodoacetate, iodoacetamide, and p-hydroxymercuribenzoate
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADP+
-
increasing NADPH/NADP+ concentration ratios inhibit the NADP-linked, but stimulate the NAD-linked reaction
NADPH
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increasing NADPH/NADP+ concentration ratios inhibit the NADP-linked, but stimulate the NAD-linked reaction
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
12
2-deoxy-D-glucose 6-phosphate
-
25C, pH 7.8, cosubstrate: NAD+
10
D-galactose 6-phosphate
-
25C, pH 7.8, cosubstrate: NAD+
0.007 - 106
D-glucose 6-phosphate
50
D-glucose 6-sulfate
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25C, pH 7.8, cosubstrate: NAD+
0.082 - 2.3
NAD+
0.0042 - 21
NADP+
200
NADP+ 2',3'-dialdehyde
-
25C, pH 7.8
0.011
thionicotinamide-NAD+
0.001
thionicotinamide-NADP+
-
pH 7.8, 25C
additional information
additional information
-
the Km-value of NAD+ is insensitive to ionic strength over the range tested but that the Km for glucose 6-phosphate is affected. This effect is greatest at pH 9.0
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.007 - 1800
D-glucose 6-phosphate
0.02 - 1423
NAD+
0.007 - 1800
NADP+
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.005 - 16250
D-glucose 6-phosphate
105
0.025 - 7031
NAD+
7
0.8 - 65330
NADP+
10
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.12
2',5'-ADP
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pH 7.8, 25C, substrate: NADP+
1.3
2'-AMP
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pH 7.8, 25C, substrate: NAD+
2
5'-AMP
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pH 7.8, 25C, substrate: NAD+
1
acetyl-CoA
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24C, pH 7.8, inhibition of NAD+-dependent reaction
36
adenosine
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pH 7.8, 25C, substrate: NAD+
3.1
ADP
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pH 7.8, 25C, substrate: NAD+
0.52
ADP-ribose
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pH 7.8, 25C, substrate: NAD+
0.5 - 1.5
ATP
0.7
CoA
-
24C, pH 7.8, inhibition of NAD+-dependent reaction
0.4
malonyl-CoA
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24C, pH 7.8, inhibition of NAD+-dependent reaction
95
N'-methylnicotinamide
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pH 7.8, 25C, substrate: NAD+
0.76 - 1.3
NAD+
0.00337 - 8.23
NADP+
1.8
NADP+ 2',3'-dialdehyde
-
25C, pH 7.8
0.006 - 0.038
NADPH
105
nicotinamide
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pH 7.8, 25C, substrate: NAD+
0.15
oleyl-CoA
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24C, pH 7.8, inhibition of NAD+-dependent reaction
0.034 - 0.04
pyridoxal 5'-diphospho-5'-adenosine
0.039
pyridoxal 5'-phosphate
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25C, pH 7.7, competitive inhibition when glucose 6-phosphate is varied at a constant, nearly saturating concentration of NAD+
additional information
vanadate
-
inhibition constants with respect to D-glucose 6-phosphate, NAD+ or NADP+
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
715
-
25C, pH 7.6, wild-type enzyme
additional information
-
a method is described which enables one to assay simultaneously the NAD+- and NADP+-linked reactions of dehydrogenases which can utilize both coenzymes. The method is based on the fact that the thionicotinamide analogs of NADH and NADPH absorb light maximally at 400 nm, a wavelength sufficiently far removed from the absorbance maximum of NADH and NADPH to permit measurements of the simultaneous reduction of NAD+ (or NADP+) and the thionicotinamide analog of NADP+ (or NAD+). Application of the method to glucosed 6-phosphate dehydrogenase from Leuconostoc mesenteroides reveals differential effects of glucose 6-phosphate concentration on the NAD+- and NADP+-linked reactions catalyzed by this enzyme which can not be detected by conventional assay procedures and which may have regulatory significance
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
log-log plots of the dependence of kcat and kcat/Km on pH for both D177N and wild-type enzyme. The kcat profile for mutant enzyme D177N shows a nearly linear increase from pH 5 until wild-type-like activity is regained at pH 10. Above pH 10 the kcat decreases precipitously. Linear regression of the data in the pH range 5-10 produces a slope of 0.9
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.7
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calculated from sequence
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
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2 * 54000
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cocrystallization of and mutant enzyme Q365, mutant enzymen S215, mutant enzyme S215 with NAD+ and mutant enzyme Q365 with NADP+, hanging-drop vapour diffusion method, structures of NADP+- and NAD+-complexed enzymes are determined at 2.2 and 2.5 A resolution
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crystallized from phosphate buffer in a form suitable for X-ray crystallographic studies. The crystals diffract to better than 2.4 A. The space group is P3(1)21 (P3(2)21), a = 105.8 A, c = 225.1 A, V = 2.18 X 10(6) A(3). The asymmetric unit probably contains a single dimer
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hanging drop method, crystallization of six cysteine-containing mutants (Q56C, S61C, S132C, S215C, Q365C, S428C) of the enzyme and the successful preparation and crystallization of a heavy atom derivative of mutant enzyme S215C
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hanging drop vapor diffusion technique, determination of the three-dimensional structure of the D177N mutant enzyme by X-ray cryocrystallography in the presence of NAD+ and in the presence of glucose 6-phosphate plus NADPH. The structure of a glucose 6-phosphate complex of a mutant (Q365C) with normal enzyme activity is also determined and substrate binding compared
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the three-dimensional structure of the H240N glucose 6-phosphate dehydrogenase is determined at 2.5 A resolution. Crystals are grown in the presence of either glucose 6-phosphate and NAD+ or glucose 6-phosphate and NADP+, hanging drop vapor diffusion method with 2.27 M ammonium sulfate
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
49
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first order rate constant for inactivation is 0.05/min. Protection by 72 mM NAD+ or by 6.3 mM glucose 6-phosphate or 72 mM NADP+
additional information
-
the enzyme is rapidly inactivated by micromolar concentrations of Fe2+ and H2O2. Fe2+ binds to the glucose 6-phosphate binding site and interaction of the enzyme-bound Fe2+ with H2O2 leads to the oxidative modification of amino acids essential for enzyme activity. Partially inactivated enzyme remains predominantly in the dimeric form, and no change in the apparent affinity of the remaining active subunits for substrate is observed. Partial inactivation leads to a decrease in the thermal stability of the remaining activity. This decrease in thermal stability could be largely overcome by the addition of glucose 6-phosphate. Thus, although exposure to H2O2 and Fe2+ results in the irreversible inactivation of the enzyme, the resulting modification is selective, leads to the formation of heterodimers of both active and inactive subunits, and does not appear to cause large scale structural changes
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
both after non-denaturing and after denaturing electrophoretic separation (SDS-PAGE) and blotting Leuconostoc mesenteroides G6PD retains its complete catalytic activity
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chymotrypsin inactivates. First order rate constant for inactivation is 0.02/min. Protection by 72 mM NAD+ or by 6.3 mM glucose 6-phosphate or 72 mM NADP+
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eluted mutant enzyme D453C shows almost double the activity of the immobilized enzyme, which is consistent with 49% activity loss due to immobilization. Mutant enzyme D205C produces a 1.8-fold higher activity compared to its immobilized state. Eluted mutant enzyme L218C shows 9.9 times the activity of its immobilized state
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pronase inactivates. First order rate constant for inactivation is 0.012/min. Protection by 72 mM NAD+ or by 6.3 mM glucose 6-phosphate or 72 mM NADP+
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thermolysin inactivates. First order rate constant for inactivation is 0.057/min. Protection by 72 mM NAD+ or by 6.3 mM glucose 6-phosphate or 72 mM NADP+
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trypsin inactivates. First order rate constant for inactivation is 0.025/min. Protection by 72 mM NAD+ or by 6.3 mM glucose 6-phosphate or 72 mM NADP+
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
guanidine-HCl
urea
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4 M. First order rate constant for inactivation is 0.019/min. Protection by 72 mM NAD+ or by 6.3 mM glucose 6-phosphate or 72 mM NADP+
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, 8 months, enzym variants L218C, D205C or D453C, no loss of activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
mutant enzymes H250N, D177N and H178N
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli strain SU294
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expression in Escherichia coli
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expression in Escherichia coli K-12 on pBR322
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D205C
-
enzyme variant with cysteine close to the dimer interface, about 30% loss of specific activity compared to wild-type enzymeshows changes in activity and the efficacy of immobilization.
D374Q
-
kcat/Km for D-glucose 6-phosphate is 7.7fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NADP+ is 7.3fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NAD+ is 12.2fold lower compared to kcat/KM of wild-type enzyme
D453C
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enzyme variant with cysteine far from the active center, no significant loss in specific activity compared to wild-type enzyme, in contrast to wild-type enzyme, the mutant enzyme is readily immobilited
H178N
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kcat/KM for D-glucose 6-phosphate (with cosubstrate NADP+) is fold lower than the value for the wild-type enzyme, kcat/KM for NADP+ is fold lower than the value for the wild-type enzyme, kcat/KM for D-glucose 6-phosphate (with cosubstrate NAD) is fold lower than the value for the wild-type enzyme, kcat/KM for D-glucose 6-phosphate (with cosubstrate NAD+) is fold lower than the value for the wild-type enzyme
H250N
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kcat/KM for D-glucose 6-phosphate (with cosubstrate NADP+) is fold lower than the value for the wild-type enzyme, kcat/KM for NADP+ is fold lower than the value for the wild-type enzyme, kcat/KM for D-glucose 6-phosphate (with cosubstrate NAD) is fold lower than the value for the wild-type enzyme, kcat/KM for D-glucose 6-phosphate (with cosubstrate NAD+) is fold lower than the value for the wild-type enzyme
K182Q
-
kcat/Km for D-glucose 6-phosphate is 127fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NADP+ is 1.1fold higher compared to kcat/KM of wild-type enzyme, kcat/Km for NAD+ is 1.4fold lower compared to kcat/KM of wild-type enzyme
K182R
-
kcat/Km for D-glucose 6-phosphate is 81fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NADP+ is 1.1fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NAD+ is 1.6fold lower compared to kcat/KM of wild-type enzyme
K21Q
-
kcat/Km for D-glucose 6-phosphate in the NADP+-dependent reaction is 76.2fold lower compared to wild-type value, kcat/Km for D-glucose 6-phosphate in the NAD+-dependent reaction is 28.5fold lower compared to wild-type value
K21R
-
kcat/Km for D-glucose 6-phosphate in the NADP+-dependent reaction is 1.9fold lower compared to wild-type value, kcat/Km for D-glucose 6-phosphate in the NAD+-dependent reaction is 1.4fold lower compared to wild-type value
K343Q
-
kcat/Km for D-glucose 6-phosphate is 445fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NADP+ is 2.7fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NAD+ is 2.6fold lower compared to kcat/KM of wild-type enzyme
K343R
-
kcat/Km for D-glucose 6-phosphate is 23fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NADP+ is 1.2fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NAD+ is 2fold lower compared to kcat/KM of wild-type enzyme
L218C
-
enzyme variant with cysteine close to the active center, about 30% loss of specific activity compared to wild-type enzyme, the mutant enzyme shows almost complete immobilization but poor carrier activity
P149G
-
kcat/Km for D-glucose 6-phosphate is 890fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NADP+ is 120fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NAD+ is 448fold lower compared to kcat/KM of wild-type enzyme
P149V
-
kcat/Km for D-glucose 6-phosphate is 5933fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NADP+ is 1265fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NAD+ is 3843fold lower compared to kcat/KM of wild-type enzyme
Q47A
-
kcat/Km for D-glucose 6-phosphate is 1.1fold higher compared to kcat/KM of wild-type enzyme, kcat/Km for NADP+ is 1.3fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NAD+ is 1.8fold lower compared to kcat/KM of wild-type enzyme
Q47E
-
kcat/Km for D-glucose 6-phosphate is 1.3fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NADP+ is 6.4fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NAD+ is 1.7fold lower compared to kcat/KM of wild-type enzyme
R46A
-
mutant glucose-6-phosphate dehydrogenases with coenzyme specificity that favors NAD+, whereas the wild-type enzyme prefers NADP+ as coenzyme; the enzyme's Km and Ki values for NADP+ are greatly increased (2-3 orders of magnitude)
R46C
-
mutant glucose-6-phosphate dehydrogenases with coenzyme specificity that favors NAD+, whereas the wild-type enzyme prefers NADP+ as coenzyme
R46E
-
kcat/Km for D-glucose 6-phosphate is 4.8fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NADP+ is 1406fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NAD+ is 6.7fold lower compared to kcat/KM of wild-type enzyme
R46Q
-
the enzyme's Km and Ki values for NADP+ are greatly increased (2-3 orders of magnitude)
T14A
-
kcat/Km for D-glucose 6-phosphate is 1.8fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NADP+ is 5.7fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NAD+ is 12.8fold lower compared to kcat/KM of wild-type enzyme
T14S
-
kcat/Km for D-glucose 6-phosphate is 1.1fold higher compared to kcat/KM of wild-type enzyme, kcat/Km for NADP+ is 1.6fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NAD+ is 1.5fold lower compared to kcat/KM of wild-type enzyme
Y179F
-
kcat/Km for D-glucose 6-phosphate is 4.9fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NADP+ is 2.2fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NAD+ is 1.7fold lower compared to kcat/KM of wild-type enzyme
Y415F
-
kcat/Km for D-glucose 6-phosphate is 1.2fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NADP+ is 1.4fold lower compared to kcat/KM of wild-type enzyme, kcat/Km for NAD+ is 1.6fold lower compared to kcat/KM of wild-type enzyme
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
denatured in 8 M urea and dissociated into its two inactive subunits (MW 50000 Da). Denaturation leads to an approximately 80% decrease in protein fluorescence and a 20-nm red shift in the emission maximum. Upon dilution, the urea-treated enzyme regains catalytic activity (approximately 70%). The reactivated enzyme is indistinguishable from the native enzyme based on a number of physicochemical and enzymological criteria. The kinetics of renaturation and reactivation are monitored. Reactivation is stimulated to different degrees by either the initial or delayed addition of NAD+, NADP+, or glucose 6-phosphate. During the initial, rapid phase of renaturation, approximately 3 of the enzyme's 12 histidine residues become unreactive toward diethyl pyrocarbonate; concomitant with the subsequent reactivation, approximately 7 more histidines become inaccessible to diethyl pyrocarbonate
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in 4 M guanidine-HCl, the dimeric enzyme glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides dissociates to subunits and is extensively unfolded. Rapid dilution of this high guanidine hydrochloride concentration allowes the enzyme to partially renature. The fraction of the enzyme which does not renature aggregates and precipitates out of solution, a process which can not be substantially prevented by stabilizing additives. A renaturation mechanism is described, which involves a bi-unimolecular (subunit association-folding) reaction sequence. This mechanism involves an inactive, dimeric, glucose-6-phosphate dehydrogenase-folding intermediate
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