Identification of a domain conferring nucleotide binding to the N-acetyl-d-glucosamine 2-epimerase (Renin binding protein).
Renin binding protein (RnBP), a cellular renin inhibitor, has been identified as the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase. Our recent studies demonstrated that rat GlcNAc 2-epimerase has a ten-times higher affinity for ATP, dATP, and ddATP than the human enzyme [Takahashi, S. et al. (2001) J. Biochem. 130, 815-821]. To identify the domain conferring nucleotide binding to GlcNAc 2-epimerase, we constructed a series of chimeric enzymes successively replacing the three domains of the human enzyme (N-terminal, middle, and C-terminal domains) with the corresponding domains of the rat enzyme. Chimeras were expressed in Escherichia coli JM109 cells under the control of the Taq promoter. The purified chimeric enzymes had GlcNAc 2-epimerase activity and inhibited renin activity in a dose-dependent manner. The recombinant human and rat enzymes required catalytic amounts of ATP with apparent K(m) values of 73 and 5.5 microM, respectively. Chimeric enzymes of HHR, RHH, and RHR (H, human type domain; R, rat type domain) had nearly the same nucleotide specificity as the human GlcNAc 2-epimerase. On the other hand, HRR, HRH, and RRH chimeras had the same nucleotide specificity as the rat enzyme. These results indicate that the middle domain of the GlcNAc 2-epimerase molecule participates in the specificity for and binding of nucleotides, and that nucleotides are essential to form the catalytic domain of the enzyme.
Takahashi
Saori
S
Department of Bioengineering, Akita Research Institute of Food and Brewing (ARIF), Sanuki, Arayamachi 010-1623, Japan. saori@arif.pref.akita.jp
Ogasawara
Hironobu
H
Takahashi
Keitaro
K
Hori
Kazuyuki
K
Saito
Kyuichi
K
Mori
Katsumi
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002238:Carbohydrate Epimerases / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry*; D020134:Catalytic Domain; D018076:DNA, Complementary / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D054306:Dideoxynucleotides; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D012083:Renin / Q000737:chemistry*; D013820:Thermolysin
D002352:Carrier Proteins; D018076:DNA, Complementary; D003838:Deoxyadenine Nucleotides; D054306:Dideoxynucleotides; D009711:Nucleotides; C037773:2',3'-dideoxyadenosine triphosphate; D000255:Adenosine Triphosphate; D012083:Renin; D013820:Thermolysin; D002238:Carbohydrate Epimerases; C024519:N-acyl-D-glucosamine 2-epimerase; C498259:RENBP protein, human; C498258:Renbp protein, rat; C026600:2'-deoxyadenosine triphosphate