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We previously reported the expression of a full-length cDNA complementary to a rat liver NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) mRNA in Escherichia coli (Q. Ma, R. Wang, C. S. Yang, and A. Y. H. Lu, 1990, Arch. Biochem. Biophys. 283, 311-317). Since cysteine residues have been suggested to be important for the catalysis of flavoproteins and a lysine residue at position 76 in NAD(P)H:quinone oxidoreductase has been proposed to be involved in electron transfer of the enzyme, we investigated the roles of lysine 76 and cysteine 179 of this enzyme in catalysis by site-directed mutagenesis. Mutant cDNA clones replacing lysine 76 with valine (K76V) and cysteine 179 with alanine (C179A) were generated by a procedure based on the polymerase chain reaction. The mutant enzymes were expressed in E. coli. The cytosolic activities of the K76V and C179A mutants were 50 and 25% of that of the wild type (DTD), due to lower levels of the mutant proteins as shown by immunoblot analysis. The mutant proteins were purified to apparent homogeneity. The purified K76V and C179A mutant enzymes maintained full activities of 2,6-dichlorophenolindophenol (DCIP) reduction compared with that of the wild type. The mutant enzymes exhibited kinetic parameters for DCIP, NADH, and NADPH similar to those of DTD except that, with K76V, the Km for NADPH was doubled. Both mutant proteins contained two molecules of FAD per enzyme molecule. Dicumarol inhibited K76V and C179A mutant activities to greater than 90% at a concentration of 10(-7) M. Heat stability studies showed that C179A was much more sensitive to inactivation at 37 degrees C than both the wild-type and K76V enzymes. It is concluded from this study that lysine 76 and cysteine 179 are not essential in catalysis and in the binding of FAD, DCIP, and dicumarol. However, lysine residue 76 appears to play a role in NADPH binding and cysteine residue 179 is important in maintaining the stability of the enzyme.
Q Ma, and
K Cui, and
R W Wang, and
A Y Lu, and
C S Yang
June 1990,
Biochemical and biophysical research communications,
Q Ma, and
K Cui, and
R W Wang, and
A Y Lu, and
C S Yang
June 2003,
Psychiatry research,
Q Ma, and
K Cui, and
R W Wang, and
A Y Lu, and
C S Yang
March 2006,
World journal of gastroenterology,
Q Ma, and
K Cui, and
R W Wang, and
A Y Lu, and
C S Yang
January 2004,
Methods in enzymology,
Q Ma, and
K Cui, and
R W Wang, and
A Y Lu, and
C S Yang
November 2019,
Chembiochem : a European journal of chemical biology,
Q Ma, and
K Cui, and
R W Wang, and
A Y Lu, and
C S Yang
March 2003,
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society,
Q Ma, and
K Cui, and
R W Wang, and
A Y Lu, and
C S Yang
October 1999,
Journal of toxicology and environmental health. Part A,
Q Ma, and
K Cui, and
R W Wang, and
A Y Lu, and
C S Yang
July 2023,
Journal of clinical biochemistry and nutrition,
Q Ma, and
K Cui, and
R W Wang, and
A Y Lu, and
C S Yang
May 1989,
The Journal of biological chemistry,
Q Ma, and
K Cui, and
R W Wang, and
A Y Lu, and
C S Yang
February 1994,
Pharmacogenetics,
