Crystallographic and kinetic investigations on the mechanism of 6-pyruvoyltetrahydropterin synthase

The enzyme 6-pyruvoyl tetrahydropterin synthase (PTPS) catalyses the second step in the de novo biosynthesis of tetrahydrobiopterin, the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin. The Zn nd Mg -dependent reaction includes a triphosphate elimination, a stereospecific r eduction of the N5-C6 double bond and the oxidation of both side-chain hydr oxyl groups. The crystal structure of the inactive mutant Cys42AlA of PTPS in complex with its natural substrate dihydroneopterinetriphosphate was det ermined at 1.9 Angstrom resolution. The active site of PTPS consists of the pterin-anchoring Glu A107 neighboured by two catalytic motifs: a Zn(II) bi nding site and an intersubunit catalytic triad formed by Cys A42, Asp B88 a nd His B89. In the free enzyme the Zn(II) is in tetravalent co-ordination w ith three histidine ligands and a water molecule. In the complex the water is replaced by the two substrate side-chain hydroxyl groups yielding a pent a-co-ordinated Zn(II) ion. the Zn(II) ion plays a crucial role in catalysis . It activates the protons of the substrate, stabilizes the intermediates a nd disfavours the breaking of the C1'C2' bond in the pyruvoyl side-chain. C ys A42 is activated by His B89 and Asp B88 for proton abstraction from the two different substrate side-chain atoms C1', and C2'. Replacing Ala A42 in the mutant structure by the wild-type Cys by modelling shows that the C1' and C2' substrate side-chain protons are at equal distances to Cys A42 S'. The basicity of Cys A42 may be increased by a catalytic triad His B89 and A sp B88. The active site of PTPS seems to be optimised to carry out proton a bstractions from two different side-chain C1' and C2' atoms, with no obviou s preference for one of them. Kinetic studies with dihydroneopterin monopho sphate reveal that the triphosphate moiety of the substrate is necessary fo r enzyme specifity. (C) 1999 Academic Press.