Identification of functionally important cysteine residues of the human renin-binding protein as the enzyme N-acetyl-D-glucosamine 2-epimerase.
Renin-binding protein (RnBP) is an endogenous renin inhibitor originally isolated from porcine kidney. It was recently identified as the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase [Takahashi, S. et al. (1999) J. Biochem. 125, 348-353] and its active site residue was determined to be cysteine 380 by site-directed mutagenesis [Takahashi, S. et al. (1999) J. Biochem. 126, 639-642]. To further investigate the relationship between structure and function of recombinant human (rh) RnBP as a GlcNAc 2-epimerase, we have constructed several C-terminal deletion and multi-cysteine/serine mutants of rhGlcNAc 2-epimerase and expressed them in Escherichia coli cells. The expression was detected by Western blotting using anti-rhRnBP antiserum. The C-terminal deletion mutant, Delta400-417, had approximately 50% activity relative to the wild-type enzyme, but other C-terminal deletion mutants, Delta380-417, Delta386-417, and Delta390-417, had no enzymatic activity. Mutational analysis of multi-cysteine/serine mutants revealed that cysteines 41 and 390 were critical for the activity or stabilization of the enzyme, while cysteine residues in the middle of the enzyme, cysteines 125, 210, 239, and 302, had no essential function in relation to the activity.
Takahashi
S
S
Department of Bioengineering, Akita Research Institute of Food and Brewing (ARIF), Sanuki, 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
Kawamura
Y
Y
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D000458:Cyanobacteria; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013552:Swine
D002352:Carrier Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D012694:Serine; D002238:Carbohydrate Epimerases; C024519:N-acyl-D-glucosamine 2-epimerase; C498259:RENBP protein, human; C498258:Renbp protein, rat; D003545:Cysteine