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

Abstract

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.(6R)-5,6,7,8-Tetrahydro-L-biopterin (BH4) 1 is an essential cofactor for phenylalanine hydroxylase (1), tyrosine hydroxylase (2), tryptophan hydroxylase (3), glycerol ether monoxygenases (4, 5), and the three isoforms of nitric-oxide synthase (6, 7). Because the activity of these enzymes is typically limited by the availability of BH4, intracellular levels of BH4 determine the rate of production of several key cell-signaling molecules including nitric oxide (NO), dopamine, norepinephrine, epinephrine, and serotonin. In mammalian cells, BH4 levels are primarily dictated by the activity of GTP cyclohydrolase I (GTPCH), the first of three enzymes in the de novo BH4 synthesis pathway (8). Mutations in the GTPCH gene are responsible for severe diseases including 3,4-dehydroxyphenylalanine (dopa)-responsive dystonia (9) and atypical phenylketonuria (10). Other neurological disorders, including Parkinson's disease (11), Alzheimer's disease (12, 13), depression (14), and vascular endothelial dysfunction resulting from diabetes (15, 16), smoking (17-19), and hypercholesterolemia (20), are all associated with deficient BH4 levels. The prototypical GTPCH inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP) has been used extensively to assess the roles of BH4 and GTPCH in physiological and pathophysiological conditions, although the exact mechanism of this inhibition has not been defined.Four decades ago, DAHP was identified as a potent inhibitor of biopterin-dependent growth of the protozoan Crithidia fasciculata (21). Investigations of biopterin excretion in rats suggested the presence of a de novo biopterin synthesis pathway that is potently inhibited following DAHP treatment (22). Subsequently, biosynthesis of 7,8-dihydrobiopterin from GTP was demons...

Biosynthesis of 7-Deazaguanosine-Modified tRNA Nucleosides: a New Role for GTP Cyclohydrolase I

“…In mammals, GCYH-I is under negative-feedback inhibition through interactions with the GCYH feedback reg- VOL. 190, 2008 PRE-Q 0 BIOSYNTHESIS 7881 on May 11, 2018 by guest http://jb.asm.org/ ulatory protein (24), but this is not observed in bacteria as they lack this protein, and the overexpression of folate or BH4 biosynthetic genes results in overproduction of the final products (52,57). In E. coli, the expression of the folE gene has been shown to be under the control of the negative regulator MetJ (30).…”

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers

“…As a rule, activities of key enzymes catalyzing the first limiting step of the anabolic pathways are regulated by allosteric feedback inhibition exerted by the product of the reaction or entire pathway. However, very little is known about the possible feedback control of RF biosynthesis at the level of GTP cyclohydrolase II, although feedback control is well documented for GTP cyclohydrolase I, which is involved in the biosynthesis of folates and tetrahydrobiopterin (44,237,283). While the structures of bacterial GTP cyclohydrolases from E. coli and B. subtilis have been studied in detail (206,257,365,373), no information on feedback inhibition of this enzyme by RF, flavin nucleotides, or other metabolites is available.…”

Direct Binding of GTP Cyclohydrolase and Tyrosine Hydroxylase

“…GTPCH Activity Assay-GTPCH activity was assayed in the presence and absence of varying concentrations of TH via microplate spectrometry as described previously (21,27).…”