Literature summary for 1.6.2.2 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.

Cloned(Commentary)

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

Protein Variants

Protein Variants	Comment	Organism
P247A	proposed NADH-binding site, soluble domain is analyzed	Sus scrofa
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
P247L	proposed NADH-binding site, soluble domain is analyzed	Sus scrofa
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
P248A	proposed NADH-binding site, soluble domain is analyzed	Sus scrofa
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
P248L	proposed NADH-binding site, soluble domain is analyzed	Sus scrofa
P248L	the mutant shows increased Km and decreased kcat values for NADH and ferricyanide compared to the wild type enzyme	Sus scrofa
P249A	proposed NADH-binding site, soluble domain is analyzed	Sus scrofa
P249A	the mutation affects the Km (NADH) values to increase slightly by a factor of 3 compared to the wild type enzyme	Sus scrofa
P249L	proposed NADH-binding site, soluble domain is analyzed	Sus scrofa
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]

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

Molecular Weight [Da]

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

Natural Substrates/ Products (Substrates)

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

Organism

Organism	UniProt	Comment	Textmining
Sus scrofa	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
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
soluble domain	Sus scrofa

Substrates and Products (Substrate)

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

Subunits

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

Synonyms

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

Turnover Number [1/s]

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

Cofactor

Cofactor	Comment	Organism	Structure
FAD	-	Sus scrofa
NADH	-	Sus scrofa

kcat/KM [mM/s]

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