Serine 19 of human 6-pyruvoyltetrahydropterin synthase is phosphorylated by cGMP protein kinase II

6-Pyruvoyltetrahydropterin synthase (PTPS) participates in tetrahydrobiopte rin cofactor biosynthesis. We previously identified in a PTPS-deficient pat ient an inactive PTPS allele with an Arg(16) to Cys codon mutation. Arg(16) is located in the protein surface exposed phosphorylation motif Arg(16)-Ar g-Ile-Ser, with Ser(19) as the putative phosphorylation site for serine-thr eonine protein kinases. Purification of recombinant PTPS-S19A from bacteria l cells resulted in an active enzyme (k(cat)/K-m = 6.4 x 10(3) M-1 s(-1)), which was similar to wild-type PTPS (k(cat)/K-m = 4.1 x 10(3) M-1 s(-1)). I n assays with purified enzymes, wild-type but not PTPS-S19A was a specific substrate for the cGMP-dependent protein kinase (cGK) type I and II. Upon e xpression in COS-1 cells, PTPS-S19A was stable but not phosphorylated and h ad a reduced activity of similar to 33% in comparison to wild-type PTPS. Ex tracts from several human cell lines, including brain, contained a kinase t hat bound to and phosphorylated immobilized wild-type, but not mutant PTPS. Addition of cGMP stimulated phosphotransferase activity 2-fold. Extracts f rom transfected COS-1 cells overexpressing cGKII stimulated Ser(19) phospho rylation more than 100-fold, but only 4-fold from cGKI overexpressing cells . Moreover, fibroblast extracts from mice! lacking cGKII exhibited signific antly reduced phosphorylation of PTPS. These results suggest that Ser(19) o f human PTPS may be a substrate for cGKII phosphorylation also in vivo, a m odification that is essential for normal activity.