Tetrahydrobiopterin-free neuronal nitric oxide synthase: Evidence for two identical highly anticooperative pteridine binding sites

被引:142
作者
Gorren, ACF
List, BM
Schrammel, A
Pitters, E
Hemmens, B
Werner, ER
Schmidt, K
Mayer, B
机构
[1] GRAZ UNIV, INST PHARMAKOL & TOXIKOL, A-8010 GRAZ, AUSTRIA
[2] UNIV INNSBRUCK, INST MED CHEM & BIOCHEM, A-6020 INNSBRUCK, AUSTRIA
关键词
D O I
10.1021/bi961931j
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The properties of neuronal nitric oxide synthase containing one tetrahydrobiopterin (BH4) per dimer [nNOS(BH4+)] were compared to those of the BH4-free enzyme [nNOS(BH4-)]. The stimulation by BH4 of the formation of L-citrulline at the expense of H2O2 production unambiguously demonstrated that BH4 is essential in coupling reductive oxygen activation to Arg oxidation. The clear difference between the Stokes radii of nNOS(BH4-) and nNOS(BH4+) indicates that the introduction of one BH4 per dimer significantly changes the enzyme structure. Whereas the heme in nNOS(BH4+) was primarily high-spin, nNOS(BH4-) contained mainly low-spin heme. This was slowly converted into the high-spin form with Arg and/or BH4, with a rate that was independent of the concentration of either compound. Dithiothreitol inhibited the Arg/BH4-induced spin conversion by stabilizing low-spin heme. Formation of high-spin heme, with rates varying from 0.04 to 0.4 min(-1), always correlated to an equally fast increase in activity. Radioligand binding studies showed the rapid association (within 20 s) of BH4 to nNOS(BH4-), but not to nNOS(BH4+), after preincubation with Arg. Complete and monophasic dissociation of radioligand occurred in the presence of excess unlabeled BH4, demonstrating the exchangeability of high-affinity bound BI-4. Studies of the association of NG-nitro-L-arginine (L-NNA) to nNOS(BH4-) revealed that excess BH4 increased the amount of bound L-NNA 2-fold. Most of the binding data are explained by a model in which nNOS dimers accommodate two identical BH4- and Arg/L-NNA-binding sites, with cooperativity between Arg- and BH4-binding and anticooperativity between the BH4-binding sites.
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页码:16735 / 16745
页数:11
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