GTP Cyclohydrolase I Inhibition by the Prototypic Inhibitor 2,4-Diamino-6-Hydroxypyrimidine

Abstract

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 GT-PCH 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.

The Mechanism of Potent GTP Cyclohydrolase I Inhibition by 2,4-Diamino-6-hydroxypyrimidine

“…4) (39). In the absence of DAHP, the K m and V max for GTP utilization were 75.52 Ϯ 9.78 M and 46.31 Ϯ 1.23 nmol H 2 NTP/min/mg protein, respectively, in accord with published values (39,43,44). On addition of DAHP, the K m for GTP was modestly increased (100.70 Ϯ 17.34 M), and V max was unchanged (47.48 Ϯ 1.89 nmol H 2 NTP/min/mg protein) as determined by nonlinear regression analysis.…”

“…L-Phe reverses this inhibition without disrupting the inhibitory complex. The apparent structural similarity of DAHP and BH4 as well as results from previous studies conducted in rat aortic smooth muscle cells (39) and in vitro (45) suggested a possible role for GFRP in the mechanism of DAHP-mediated inhibition. Based on careful analyses using pure recombinant proteins, our findings confirm that a major mode of DAHP inhibition of GTPCH is indeed dependent on GFRP.…”

“…Expression and Purification of Recombinant GTPCH-Recombinant rat GTPCH was expressed as a maltose-binding protein (MBP) fusion protein and purified from E. coli as described previously (39). In brief, DH5␣ E. coli were transformed with a pMAL-P2 expression plasmid engineered to express GTPCH-MBP fusion protein.…”

“…1), it was considered that DAHP may be acting as a BH4-mimetic to engage GFRP-mediated feedback inhibition of GTPCH (39). To test this possibility, recombinant GFRP was purified from a prokaryotic expression system (Fig.…”

“…Because L-Phe is potentially neurotoxic, the ability of L-Phe to stimulate GTPCH activity may have evolved to provide an abundant supply of BH4 cofactor for optimal activity of phenylalanine hydroxylase. A previous observation that DAHP-mediated inhibition of GTPCH in rat aortic smooth muscle cells is partially reversed by addition of L-Phe suggests that the mode of inhibition by DAHP may be GFRP-dependent (39). Nonetheless, DAHP has been widely assumed to selectively inhibit GTPCH activity via competition for binding substrate GTP.…”