Identification of cysteine-380 as the essential residue for the human N-acetyl-D-glucosamine 2-epimerase (renin binding protein).
Renin binding protein (RnBP) is a proteinous renin inhibitor firstly isolated from porcine kidney. Recently, the protein was identified as the enzyme, N-acetyl-D-glucosamine (GlcNAc) 2-epimerase. The GlcNAc 2-epimerase activity of recombinant human RnBP was specifically inhibited by SH-reagents such as N-ethylmaleimide, 5, 5'-dithiobis-2-nitrobenzoate, and iodoacetic acid, indicating that the most probable reactive site is a cysteine residue. To identify the active site residue(s), we have constructed ten cysteine residue mutants (C41S, C66S, C104S, C125S, C210S, C239S, C302S, C380S, C386S, and C390S) for human GlcNAc 2-epimerase and expressed them in Escherichia coli cells. The relative specific activities of C41S, C66S, C125S, C210S, C239S, C302S, C386S, and C390S are nearly the same to that of the wild-type enzyme. The specific activity of the C104S mutant is 26% of that of the wild-type enzyme. The expression of the C380S mutant in E. coli cells was detected on Western blotting, whereas GlcNAc 2-epimerase activity was not detected in the extract. These results indicate that Cys380 is essential for the enzymatic activity of human GlcNAc 2-epimerase.
Takahashi
S
S
Department of Bioengineering, Akita Research Institute of Food and Brewing (ARIF), Arayamachi, Akita, 010-1623, Japan. saori@arif.pref. akita.jp
Takahashi
K
K
Kaneko
T
T
Ogasawara
H
H
Shindo
S
S
Saito
K
K
Kobayashi
M
M
D000818:Animals; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D002352:Carrier Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D002238:Carbohydrate Epimerases; C024519:N-acyl-D-glucosamine 2-epimerase; C498259:RENBP protein, human; D003545:Cysteine