The mechanism of potent GTP cyclohydrolase I inhibition by 2,4-diamino-6-hydroxypyrimidine: requirement of the GTP cyclohydrolase I feedback regulatory protein.
Inhibition of GTP cyclohydrolase I (GTPCH) has been used as a selective tool to assess the role of de novo synthesis of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) in a biological system. Toward this end, 2,4-diamino-6-hydroxypyrimidine (DAHP) has been used as the prototypical GTPCH inhibitor. Using a novel real-time kinetic microplate assay for GTPCH activity and purified prokaryote-expressed recombinant proteins, we show that potent inhibition by DAHP is not the result of a direct interaction with GTPCH. Rather, inhibition by DAHP in phosphate buffer occurs via an indirect mechanism that requires the presence of GTPCH feedback regulatory protein (GFRP). Notably, GFRP was previously discovered as the essential factor that reconstitutes inhibition of pure recombinant GTPCH by the pathway end product BH4. Thus, DAHP inhibits GTPCH by engaging the endogenous feedback inhibitory system. We further demonstrate that L-Phe fully reverses the inhibition of GTPCH by DAHP/GFRP, which is also a feature in common with inhibition by BH4/GFRP. These findings suggest that DAHP is not an indiscriminate inhibitor of GTPCH in biological systems; instead, it is predicted to preferentially attenuate GTPCH activity in cells that most abundantly express GFRP and/or contain the lowest levels of L-Phe.
Department of Pharmacology, Weill Medical College of Cornell University, New York, New York 10021, USA.
Monica A
MA
Kolinsky
Steven S
SS
Gross
D000818:Animals; D001011:Aorta / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006136:GTP Cyclohydrolase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007042:Hypoxanthines / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; C491065:Gchfr protein, rat; D007042:Hypoxanthines; D047908:Intracellular Signaling Peptides and Proteins; D010710:Phosphates; D011506:Proteins; D011994:Recombinant Proteins; D010649:Phenylalanine; D006136:GTP Cyclohydrolase; C017202:2,4-diaminohypoxanthine