Location of the active site and proposed catalytic mechanism of pterin-4a-carbinolamine dehydratase.
Based on the recently solved three-dimensional structure of pterin-4a-carbinolamine dehydratase from rat/human liver the involvement of the proposed active-site residues Glu57, Asp60, His61, His62, Tyr69, His79, Arg87 and Asp88 was examined by site-directed mutagenesis. Most of the mutants showed reduced activity, and only the Glu57-->Ala mutant and the His61-->Ala, His62-->Ala double mutant were fully devoid of activity. The dissociation constants of quinonoid 6,6-dimethyl-7,8-dihydropterin were significantly increased for binding to the Glu57-->Ala, His61-->Ala, His62-->Ala single mutants and the His61-->Ala, His62-->Ala double mutant, confirming that His61 and His62 are essential for substrate binding and catalysis. The mechanism of dehydration is proposed to involve base catalysis at the N(5)-H group of the substrate by His61.
Köster
S
S
Department of Biology, University of Konstanz, Germany.
Stier
G
G
Ficner
R
R
Hölzer
M
M
Curtius
H C
HC
Suck
D
D
Ghisla
S
S
D000595:Amino Acid Sequence; D001665:Binding Sites; D018698:Glutamic Acid / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006801:Humans; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011621:Pteridines / Q000378:metabolism; D011809:Quinones / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D011621:Pteridines; D011809:Quinones; D011994:Recombinant Proteins; D014157:Transcription Factors; D018698:Glutamic Acid; D006639:Histidine; D006836:Hydro-Lyases; C076392:pterin-4a-carbinolamine dehydratase