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 first and rate-limiting enzyme in the pathway for synthesis of tetrahydrobiopterin (BH4). Accordingly, DAHP has been widely employed to distinguish whether de novo BH4 synthesis is required in a given biological system. Although it has been assumed that DAHP inhibits GTPCH by direct competition with substrate GTP, this has never been formally demonstrated. 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 by reverse transcription-polymerase chain reaction that GFRP mRNA is constitutively expressed in rat aortic smooth muscle cells and further induced by treatment with immunostimulants. Moreover, functional GFRP is expressed and immunostimulant-induced BH4 accumulates in sufficient quantity to trigger feedback inhibition of GTPCH. Studies with DAHP reveal 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 feedback inhibitory system; at higher concentrations, DAHP competes directly for binding with GTP substrate. This knowledge predicts that DAHP would preferably target GTPCH in tissues with abundant GFRP.
Department of Pharmacology, Cornell University Medical College, New York, New York 10021, USA.
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D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006136:GTP Cyclohydrolase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D007042:Hypoxanthines / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology; D010649:Phenylalanine / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats
D017931:DNA Primers; D004791:Enzyme Inhibitors; C491065:Gchfr protein, rat; D007042:Hypoxanthines; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D012333:RNA, Messenger; D010649:Phenylalanine; D006136:GTP Cyclohydrolase; C017202:2,4-diaminohypoxanthine