GTP CYCLOHYDROLASE-I INHIBITION BY THE PROTOTYPIC INHIBITOR 2,4-DIAMINO-6-HYDROXYPYRIMIDINE - MECHANISMS AND UNANTICIPATED ROLE OF GTP CYCLOHYDROLASE-I FEEDBACK REGULATORY PROTEIN

2,4-Diamino-6-hydroxypyrimidine (DAHP) is considered to be a selective and direct-acting inhibitor of GTP cyclohydrolase I (GTPCH), the firs t and rate-limiting enzyme in the pathway for synthesis of tetrahydrob iopterin (BH4). Accordingly, DAHP has been widely employed to distingu ish whether de novo BH4 synthesis is required in a given biological sy stem. Although it has been assumed that DAHP inhibits GTPCH by direct competition with substrate GTP, this has never been formally demonstra ted. In view of apparent structural homology between DAHP and BH4, we questioned whether DAHP may mimic BH4 in its inhibition of GTPCH by an indirect mechanism, involving interaction with a recently cloned 9.5- kDa protein termed GTPCH Feedback Regulatory Protein (GFRP). We show b y reverse transcription-polymerase chain reaction that GFRP mRNA is co nstitutively expressed in rat aortic smooth muscle cells and further i nduced by treatment with immunostimulants. Moreover, functional GFRP i s expressed and immunostimulant-induced BH4 accumulates in sufficient quantity to trigger feedback inhibition of GTPCH. Studies with DAHP re veal that GFRP is also essential to achieve potent inhibition of GTPCH . Indeed, DAHP inhibits GTPCH by dual mechanisms. At a relatively low concentration DAHP emulates BH4 and engages the GFRP-dependent feedbac k inhibitory system; at higher concentrations, DAHP competes directly for binding with GTP substrate. This knowledge predicts that DAHP woul d preferably target GTPCH in tissues with abundant GFRP.