Nishimura J S, Martasek P, McMillan K, Salerno J, Liu Q, Gross S S, Masters B S
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760, USA.
Biochem Biophys Res Commun. 1995 May 16;210(2):288-94. doi: 10.1006/bbrc.1995.1659.
A putative dihydrofolate reductase (DHFR) module has been identified in neuronal nitric oxide synthase, consisting of amino acids 558-721, and is proposed to be the site of tetrahydrobiopterin (BH4) binding. This polypeptide has been expressed in E. coli as a fusion protein with glutathione S-transferase (GST), using the plasmid pGEX-4T1. The protein binds N omega-nitro-L-arginine (NNA) tightly, but this binding is not stimulated by BH4. cDNAs for Module II (residues 220-557) and Module III (residues 220-721) have been expressed as fusion proteins with GST. Module II does not bind NNA. However, Module III does bind NNA and binding is significantly stimulated by BH4. These observations are taken as strong evidence that the DHFR module contains the L-arginine binding site and, presumably, the BH4 binding site by analogy to its homology with DHFR, but that tight binding of BH4 requires amino acids 220-577.
在神经元型一氧化氮合酶中已鉴定出一个假定的二氢叶酸还原酶（DHFR）模块，其由氨基酸558 - 721组成，并被认为是四氢生物蝶呤（BH4）的结合位点。使用质粒pGEX - 4T1，该多肽已在大肠杆菌中作为与谷胱甘肽S - 转移酶（GST）的融合蛋白表达。该蛋白紧密结合Nω-硝基 - L - 精氨酸（NNA），但这种结合不受BH4刺激。模块II（残基220 - 557）和模块III（残基220 - 721）的cDNA已作为与GST的融合蛋白表达。模块II不结合NNA。然而，模块III确实结合NNA，并且结合受到BH4的显著刺激。这些观察结果被视为强有力的证据，即DHFR模块包含L - 精氨酸结合位点，并且大概通过与DHFR的同源性包含BH4结合位点，但BH4的紧密结合需要氨基酸220 - 577。
