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Literature summary extracted from

  • Nishimura, Y.; Shibuya, M.; Muraki, A.; Takeuchi, F.; Park, S.; Tsubaki, M.
    Structural and mechanistic roles of three consecutive Pro residues of porcine NADH-cytochrome b5 reductase for the binding of beta-NADH (2009), J. Biosci. Bioeng., 108, 286-292.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.6.2.2 expressed in Escherichia coli BL21(DE3) cells Sus scrofa
1.6.2.2 soluble domain, His-tag Sus scrofa

Protein Variants

EC Number Protein Variants Comment Organism
1.6.2.2 P247A proposed NADH-binding site, soluble domain is analyzed Sus scrofa
1.6.2.2 P247A the mutant shows increased Km and decreased kcat values for NADH and cytochrome b5, as well as increased Km and kcat values for ferricyanide compared to the wild type enzyme Sus scrofa
1.6.2.2 P247L proposed NADH-binding site, soluble domain is analyzed Sus scrofa
1.6.2.2 P247L the mutation significantly decreases kcat with slight increase (about 2fold) in Km for the physiological electron donor NADH. However, Km and kcat values for the electron acceptors (both cytochrome b5 and ferricyanide) are decreased significantly Sus scrofa
1.6.2.2 P248A proposed NADH-binding site, soluble domain is analyzed Sus scrofa
1.6.2.2 P248A the mutant shows increased Km and decreased kcat values for NADH and cytochrome b5, as well as increased Km and kcat values for ferricyanide compared to the wild type enzyme Sus scrofa
1.6.2.2 P248L proposed NADH-binding site, soluble domain is analyzed Sus scrofa
1.6.2.2 P248L the mutant shows increased Km and decreased kcat values for NADH and ferricyanide compared to the wild type enzyme Sus scrofa
1.6.2.2 P249A proposed NADH-binding site, soluble domain is analyzed Sus scrofa
1.6.2.2 P249A the mutation affects the Km (NADH) values to increase slightly by a factor of 3 compared to the wild type enzyme Sus scrofa
1.6.2.2 P249L proposed NADH-binding site, soluble domain is analyzed Sus scrofa
1.6.2.2 P249L the mutant shows increased Km and kcat values for NADH, ferricyanide and cytochrome b5 compared to the wild type enzyme Sus scrofa

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.6.2.2 0.00104
-
ferricyanide mutant P247L Sus scrofa
1.6.2.2 0.00104
-
ferricyanide mutant enzyme P247L, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 0.00693
-
ferricytochrome b5 mutant P247L Sus scrofa
1.6.2.2 0.00723
-
ferricyanide
-
Sus scrofa
1.6.2.2 0.00723
-
ferricyanide wild type enzyme, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 0.01096
-
ferricyanide mutant P247A Sus scrofa
1.6.2.2 0.01096
-
ferricyanide mutant enzyme P247A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 0.01097
-
NADH ferricyanide as electron acceptor Sus scrofa
1.6.2.2 0.01097
-
NADH wild type enzyme, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 0.0135
-
NADH mutant P248L, ferricyanide as electron acceptor Sus scrofa
1.6.2.2 0.0135
-
NADH mutant enzyme P248L, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 0.01353
-
ferricytochrome b5 wild type enzyme, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 0.0155
-
NADH mutant P249L, ferricyanide as electron acceptor Sus scrofa
1.6.2.2 0.0155
-
NADH mutant enzyme P249L, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 0.01563
-
ferricyanide mutant P248A Sus scrofa
1.6.2.2 0.01563
-
ferricyanide mutant enzyme P248A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 0.01568
-
ferricyanide mutant P248L Sus scrofa
1.6.2.2 0.01568
-
ferricyanide mutant enzyme P248L, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 0.01713
-
NADH mutant P247A, ferricyanide as electron acceptor Sus scrofa
1.6.2.2 0.01713
-
NADH mutant enzyme P247A, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 0.01735
-
ferricytochrome b5
-
Sus scrofa
1.6.2.2 0.0207
-
ferricytochrome b5 mutant enzyme P247A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 0.02407
-
ferricytochrome b5 mutant P247A Sus scrofa
1.6.2.2 0.02407
-
NADH mutant P248A, ferricyanide as electron acceptor Sus scrofa
1.6.2.2 0.02407
-
NADH mutant enzyme P248A, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 0.02467
-
NADH mutant P247L, ferricyanide as electron acceptor Sus scrofa
1.6.2.2 0.02467
-
NADH mutant enzyme P247L, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 0.02646
-
ferricyanide mutant P249A Sus scrofa
1.6.2.2 0.02646
-
ferricyanide mutant enzyme P249A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 0.02928
-
NADH mutant P249A, ferricyanide as electron acceptor Sus scrofa
1.6.2.2 0.02928
-
NADH mutant enzyme P249A, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 0.03309
-
ferricyanide mutant P249L Sus scrofa
1.6.2.2 0.03309
-
ferricyanide mutant enzyme P249L, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 0.03434
-
ferricytochrome b5 mutant enzyme P248A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 0.04148
-
ferricytochrome b5 mutant P248A Sus scrofa
1.6.2.2 0.04481
-
ferricytochrome b5 mutant enzyme P249A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 0.05238
-
ferricytochrome b5 mutant P249A Sus scrofa
1.6.2.2 0.055
-
ferricytochrome b5 mutant P249L Sus scrofa
1.6.2.2 0.08199
-
ferricytochrome b5 mutant P248L Sus scrofa

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.6.2.2 30810
-
calculated from amino acid sequence Sus scrofa
1.6.2.2 31000
-
SDS-PAGE Sus scrofa
1.6.2.2 33000
-
x * 33000 (soluble domain), SDS-PAGE Sus scrofa

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.6.2.2 2 ferricytochrome b5 + NADH Sus scrofa
-
2 ferrocytochrome b5 + NAD+ + H+
-
?

Organism

EC Number Organism UniProt Comment Textmining
1.6.2.2 Sus scrofa
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.6.2.2 ammonium sulfate precipitation, DEAE-Sepharose CL-6B column chromatography, 5'-ADP-agarose (or 5'-AMP-agarose) gel column chromatography, and Ni-NTA-agarose column chromatography Sus scrofa
1.6.2.2 soluble domain Sus scrofa

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.6.2.2 2 ferricyanide + NADH
-
Sus scrofa 2 ferrocyanide + NAD+ + H+
-
?
1.6.2.2 2 ferricytochrome b5 + NADH
-
Sus scrofa 2 ferrocytochrome b5 + NAD+ + H+
-
?
1.6.2.2 NADH + ferricytochrome b5
-
Sus scrofa NAD+ + H+ + ferrocytochrome b5
-
r

Subunits

EC Number Subunits Comment Organism
1.6.2.2 ? x * 33000 (soluble domain), SDS-PAGE Sus scrofa

Synonyms

EC Number Synonyms Comment Organism
1.6.2.2 B5R
-
Sus scrofa
1.6.2.2 NADH-cytochrome b5 reductase
-
Sus scrofa
1.6.2.2 NADH-ferricyanide reductase
-
Sus scrofa
1.6.2.2 NADH-ferricytochrome reductase
-
Sus scrofa
1.6.2.2 NADH:cytochrome b5 reductase
-
Sus scrofa

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.6.2.2 21.7
-
NADH mutant P247L, ferricyanide as electron acceptor Sus scrofa
1.6.2.2 21.7
-
NADH mutant enzyme P247L, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 27.2
-
ferricytochrome b5 mutant P247L Sus scrofa
1.6.2.2 30.8
-
ferricyanide mutant P247L Sus scrofa
1.6.2.2 30.8
-
ferricyanide mutant enzyme P247L, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 147
-
ferricytochrome b5 wild type enzyme, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 167
-
ferricytochrome b5
-
Sus scrofa
1.6.2.2 167
-
ferricytochrome b5 mutant enzyme P247A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 200
-
ferricytochrome b5 mutant P247A Sus scrofa
1.6.2.2 270
-
ferricytochrome b5 mutant P249L Sus scrofa
1.6.2.2 275
-
ferricytochrome b5 mutant P248L Sus scrofa
1.6.2.2 288
-
ferricytochrome b5 mutant P248A Sus scrofa
1.6.2.2 301
-
ferricytochrome b5 mutant enzyme P248A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 337
-
ferricyanide
-
Sus scrofa
1.6.2.2 337
-
ferricyanide wild type enzyme, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 354
-
NADH mutant P247A, ferricyanide as electron acceptor Sus scrofa
1.6.2.2 354
-
NADH mutant enzyme P247A, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 357
-
ferricyanide mutant P247A Sus scrofa
1.6.2.2 357
-
ferricyanide mutant enzyme P247A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 399
-
ferricyanide mutant P248A Sus scrofa
1.6.2.2 399
-
ferricyanide mutant enzyme P248A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 441
-
ferricytochrome b5 mutant P249A Sus scrofa
1.6.2.2 504
-
ferricyanide mutant P248L Sus scrofa
1.6.2.2 504
-
ferricyanide mutant enzyme P248L, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 540
-
NADH mutant P248L, ferricyanide as electron acceptor Sus scrofa
1.6.2.2 540
-
NADH mutant enzyme P248L, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 540
-
ferricytochrome b5 mutant enzyme P249A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 559
-
NADH mutant P248A, ferricyanide as electron acceptor Sus scrofa
1.6.2.2 559
-
NADH mutant enzyme P248A, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 681
-
NADH mutant P249A, ferricyanide as electron acceptor Sus scrofa
1.6.2.2 681
-
NADH mutant enzyme P249A, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 742
-
NADH ferricyanide as electron acceptor Sus scrofa
1.6.2.2 742
-
NADH wild type enzyme, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 838
-
ferricyanide mutant P249A Sus scrofa
1.6.2.2 838
-
ferricyanide mutant enzyme P249A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 1059
-
NADH mutant P249L, ferricyanide as electron acceptor Sus scrofa
1.6.2.2 1059
-
NADH mutant enzyme P249L, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 1241
-
ferricyanide mutant P249L Sus scrofa
1.6.2.2 1241
-
ferricyanide mutant enzyme P249L, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa

Cofactor

EC Number Cofactor Comment Organism Structure
1.6.2.2 FAD
-
Sus scrofa
1.6.2.2 NADH
-
Sus scrofa

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.6.2.2 880
-
NADH mutant enzyme P247L, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 20670
-
NADH mutant enzyme P247A, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 23220
-
NADH mutant enzyme P248A, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 23260
-
NADH mutant enzyme P249A, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 25530
-
ferricyanide mutant enzyme P248A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 29620
-
ferricyanide mutant enzyme P247L, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 31670
-
ferricyanide mutant enzyme P249A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 32140
-
ferricyanide mutant enzyme P248L, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 32570
-
ferricyanide mutant enzyme P247A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 37500
-
ferricyanide mutant enzyme P249L, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 40000
-
NADH mutant enzyme P248L, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 46610
-
ferricyanide wild type enzyme, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 67640
-
NADH wild type enzyme, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 68320
-
NADH mutant enzyme P249L, using ferricyanide as cosubstrate, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 80700
-
ferricytochrome b5 mutant enzyme P247A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 87700
-
ferricytochrome b5 mutant enzyme P248A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 108600
-
ferricytochrome b5 wild type enzyme, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa
1.6.2.2 120500
-
ferricytochrome b5 mutant enzyme P249A, in 100 mM potassium phosphate buffer (pH 7.0) at 25°C Sus scrofa