Identification of the essential cysteinyl residue located in the active site of human phenylethanolamine N-methyltransferase.
Phenylethanolamine N-methyltransferase (PNMT) catalyzes the production of epinephrine from norepinephrine using S-adenosyl-L-methionine as a methyl donor. Previous studies of chemical modification of the PNMT with reagents specific to Cys residues showed that the enzyme contains a Cys residue essential for its activity. Each of the six Cys residues in human PNMT was changed to Ser by PCR-based site-directed mutagenesis, and each mutant PNMT was expressed in Escherichia coli to identify the functionally important Cys residue. The six mutants (C48S, C60S, C91S, C131S, C139S, and C183S) and the wild-type enzyme were expressed at almost the same levels as revealed by Western blotting analysis. Kinetic parameters (apparent Km and Vmax) of C48S, C60S, C91S, C131S, and C139S for the substrates, norepinephrine and S-adenosyl-L-methionine, showed similar values to those of the wild-type enzyme. However, C183S exhibited markedly reduced enzyme activity with less than 3% of the wild-type Vmax and with ca. sixfold increased apparent Km values for both substrates. These results suggested that Cys183 plays an important role in the activity of human PNMT.
Kaneda
N
N
Faculty of Pharmacy, Meijo University, Nagoya, 468-8503, Japan. nkaneda@meijo-u.ac.jp
Hikita
K
K
Naruse
Y
Y
Fukuo
T
T
Matsubara
K
K
Nagatsu
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003545:Cysteine / Q000032:analysis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009638:Norepinephrine / Q000378:metabolism; D010625:Phenylethanolamine N-Methyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012436:S-Adenosylmethionine / Q000378:metabolism; D017385:Sequence Homology; D012694:Serine / Q000235:genetics; D013379:Substrate Specificity
D012694:Serine; D012436:S-Adenosylmethionine; D010625:Phenylethanolamine N-Methyltransferase; D003545:Cysteine; D009638:Norepinephrine