STRUCTURAL AND FUNCTIONAL CONSEQUENCES OF MUTATIONS IN 6-PYRUVOYLTETRAHYDROPTERIN SYNTHASE CAUSING HYPERPHENYLALANINEMIA IN HUMANS - PHOSPHORYLATION IS A REQUIREMENT FOR IN-VIVO ACTIVITY

Four naturally occurring mutants with single amino acid alterations in human 6-pyruvoyltetrahydropterin synthase (PTPS) were overexpressed a nd characterized in vitro. The corresponding DNA mutations were found in patients with hyperphenylalaninemia and monoamine neurotransmitter insufficiency due to lack of the tetrahydrobiopterin biosynthetic enzy me PTPS. To predict the structure of the mutant enzymes, computer mode ling was performed based on the solved three-dimensional structure of the homohexameric rat enzyme. One mutant (Delta V57) is incorrectly fo lded and thus unstable in vitro and in vivo, while a second mutant (P8 7L) has substantial activity but enhanced sensitivity to local unfoldi ng. Two other mutants, R16C and R25Q, form stable homomultimers and ex hibit significant activity in vitro but no activity in COS-1 cells, In vivo P-32 labeling showed that nild-type PTPS, P87L, and R25Q are pho sphorylated, while R16C is not modified. This strongly suggests that t he serine 19 within the consensus sequence for various kinases, RXXS, is the site of modification, Our results demonstrate that PTPS undergo es protein phosphorylation and requires additional, not yet identified post-translational modification(s) for its in vivo function.