TETRAHYDROBIOPTERIN BINDING TO MACROPHAGE INDUCIBLE NITRIC-OXIDE SYNTHASE - HEME SPIN SHIFT AND DIMER STABILIZATION BY THE POTENT PTERIN ANTAGONIST 4-AMINO-TETRAHYDROBIOPTERIN
B. Mayer et al., TETRAHYDROBIOPTERIN BINDING TO MACROPHAGE INDUCIBLE NITRIC-OXIDE SYNTHASE - HEME SPIN SHIFT AND DIMER STABILIZATION BY THE POTENT PTERIN ANTAGONIST 4-AMINO-TETRAHYDROBIOPTERIN, Biochemistry, 36(27), 1997, pp. 8422-8427
The characteristics of tetrahydrobiopterin (H(4)biopterin) binding to
pteridine-free recombinant macrophage inducible nitric oxide synthase
expressed in Escherichia call were investigated with a special focus g
iven to effects caused by trhydro-6-(L-erythro-1,2-dihydroxypropyl)pte
ridine (4-amino-H(4)biopterin), a novel pterin-based inhibitor of nitr
ic oxide synthase. The 4-amino compound completely inhibited enzyme st
imulation by 10 mu M H(4)biopterin with a half-maximally active concen
tration of 7.2 +/- 0.39 mu M, whereas H(2)biopterin and sepiapterin we
re much less potent. Binding studies using [H-3]H(4)biopterin at 4 deg
rees C revealed biphasic association of the radioligand according to t
wo first-order reactions with apparent rate constants of 2.2 and 0.05
min(-1), each accounting for approximately 50% of total binding. Disso
ciation of [H-3]H(4)biopterin occurred with rate constants of 0.005 an
d 0.0028 min(-1) in the absence and presence of L-arginine, respective
ly. Specific binding of 10 nM [H-3]H(4)biopterin was antagonized by un
labeled H(4)biopterin and its 4-amino analog with half-maximal effects
at 84 +/- 6 and 34 +/- 3.2 nM, respectively. Binding of H(4)biopterin
and 4-amino-H(4)biopterin was accompanied by a partial low spin to hi
gh spin conversion of the heme that was completed by L-arginine, Simil
arly, the active cofactor and the inhibitory 4-amino derivative both i
nduced significant formation of stable protein dimers that survived du
ring SDS electrophoresis, suggesting that the allosteric effects cause
d by H(4)biopterin do not explain sufficiently the essential role of t
he pteridine cofactor in NO biosynthesis.