Cassette mutagenesis and photoaffinity labeling of adenine binding domain of ADP regulatory site within human glutamate dehydrogenase.
The adenine binding domain of the ADP site within human glutamate dehydrogenase (GDH) was identified by cassette mutagenesis at the Tyr187 position. The wild type GDH was activated 3-fold by ADP at a concentration of 1 mM at pH 8.0, whereas no significant activation by ADP was observed with the Tyr187 mutant GDH regardless of the size, hydrophobicity, and ionization of the side chains. Studies of the steady-state velocity of the mutant enzymes revealed essentially unchanged apparent K(m) values for 2-oxoglutarate and NADH, but an approximately 4-fold decrease in the respective apparent V(max) values. The binding of ADP to the wild type or mutant GDH was further examined by photoaffinity labeling with [alpha-(32)P]8-azidoadenosine 5'-diphosphate (8N(3)ADP). 8N(3)ADP, without photolysis, mimicked the stimulatory properties of ADP on GDH activity. Saturation of photoinsertion with 8N(3)ADP occurred with apparent K(d) values near 25 microM for the wild type GDH, and the photoinsertion of [alpha-(32)P]8N(3)ADP was decreased best by ADP in comparison to other nucleotides. Unlike the wild type GDH, essentially no photoinsertion was detected for the Tyr187 mutant GDH in the presence or absence of 1 mM ADP. For the wild type GDH, photolabel-containing peptide generated by tryptic digestion was identified in the region containing the sequence EMSWIADTYASTIG, and the photolabeling of this peptide was prevented >95% by the presence of 1 mM ADP during photolysis, whereas no such a peptide was detected for the Tyr187 mutant GDH in the presence or absence of ADP. These results with cassette mutagenesis and photoaffinity labeling demonstrate selectivity of the photoprobe for the ADP binding site and suggest that the photolabeled peptide is within the ADP binding domain of the human GDH and that Tyr187 is responsible for the efficient base binding of ADP to human GDH.
Yoon
Hye-Young
HY
Department of Biochemistry, University of Ulsan College of Medicine, 388-1 Poongnap-Dong, Songpa-Ku, Seoul 138-736, South Korea.
Lee
Eun-Young
EY
Cho
Sung-Woo
SW
D000225:Adenine / Q000378:metabolism*; D000241:Adenosine / Q000031:analogs & derivatives* / Q000032:analysis; D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000032:analysis; D001665:Binding Sites; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008322:Mammals; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D019920:Photoaffinity Labels / Q000032:analysis; D017434:Protein Structure, Tertiary; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
D001386:Azides; D010455:Peptides; D019920:Photoaffinity Labels; D014443:Tyrosine; C012760:8-azidoadenosine; D000244:Adenosine Diphosphate; D005969:Glutamate Dehydrogenase; D000225:Adenine; D000241:Adenosine