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 t he mutants showed reduced activity, and only the Glu57-->Ala mutant an d the His61-->Ala. His62-->Ala double mutant were fully devoid of acti vity. The dissociation constants of quinonoid 6,6-dimethyl-7,8-dihydro pterin were significantly increased for binding to the Glu57-->Ala, Hi s61-->Ala. His62-->Ala single mutants and the His61-->Ala, His62-->Ala double mutant, confirming that His61 and His62 are essential for subs trate binding and catalysis. The mechanism of dehydration is proposed to involve base catalysis at the N(5)-H group of the substrate by His6 1.