Groth G, Mills D A, Christiansen E, Richter M L, Huchzermeyer B
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, USA.
Biochemistry. 2000 Nov 14;39(45):13781-7. doi: 10.1021/bi000991t.
The photoaffinity phosphate analogue 4-azido-2 nitrophenyl phosphate (ANPP) was shown previously (Pougeois, R., Lauquin, G. J.-M., and Vignais, P. V. (1983) Biochemistry 22, 1241-1245) to bind covalently and specifically to a single catalytic site on one of the three beta-subunits of the isolated chloroplast coupling factor 1 (CF(1)). Modification by ANPP strongly inhibited ATP hydrolysis activity. In this study, we examined labeling of membrane-bound CF(1) by ANPP by exposing thylakoid membranes to increasing concentrations of the reagent. ANPP exhibited saturable binding to two sites on CF(1), one on the beta-subunit and one on the alpha-subunit. Labeling by ANPP resulted in the complete inhibition of both ATP synthesis and ATP hydrolysis by the membrane-bound enzyme. Labeling of both sites by ANPP was reduced by more than 80% in the presence of P(i) (> or = 10 mM) and ATP (> or = 0.5 mM). ADP was less effective in competing with ANPP for binding, giving a maximum of approximately 35% inhibition at concentrations > or = 2 mM. ANPP-labeled tryptic peptides of the alpha-subunit were isolated and sequenced. The majority of the probe was contained in three peptides corresponding to residues Gln(173) to Arg(216), Gly(217) to Arg(253), and His(256) to Arg(272) of the alpha-subunit. In the mitochondrial F(1) (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), all three analogous peptides are located within the nucleotide binding pocket and within close proximity to the gamma-phosphate binding site. The data indicate, however, that the azidophenyl group of bound ANPP is oriented at approximately 180 degrees in the opposite direction to the adenine binding site with reference to the phosphate binding site on the alpha-subunit. The study has confirmed that ANPP is a bona fide phosphate analogue and suggests that it specifically targets the gamma-phosphate binding site within the nucleotide binding pockets on the alpha- and beta-subunits of CF(1). The study also indicates that in the resting state of the chloroplast F(1)-F(0) complex both the alpha- and beta-subunits are structurally asymmetric.
光亲和性磷酸类似物4-叠氮基-2-硝基苯磷酸酯（ANPP）先前已被证明（Pougeois, R., Lauquin, G. J.-M., and Vignais, P. V. (1983) Biochemistry 22, 1241-1245）能共价且特异性地结合到分离的叶绿体偶联因子1（CF(1)）三个β亚基之一上的单个催化位点。ANPP修饰强烈抑制ATP水解活性。在本研究中，我们通过将类囊体膜暴露于浓度不断增加的该试剂中来检测ANPP对膜结合CF(1)的标记。ANPP对CF(1)上的两个位点表现出饱和结合，一个在β亚基上，一个在α亚基上。ANPP标记导致膜结合酶的ATP合成和ATP水解完全受到抑制。在存在Pi（≥10 mM）和ATP（≥0.5 mM）的情况下，ANPP对两个位点的标记减少了80%以上。ADP在与ANPP竞争结合方面效果较差，在浓度≥2 mM时最大抑制约为35%。分离并测序了ANPP标记的α亚基的胰蛋白酶肽段。大部分探针包含在对应于α亚基Gln(173)至Arg(216)、Gly(217)至Arg(253)和His(256)至Arg(272)残基的三个肽段中。在线粒体F(1)中（Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628），所有三个类似肽段都位于核苷酸结合口袋内且紧邻γ-磷酸结合位点。然而，数据表明，结合的ANPP的叠氮苯基相对于α亚基上的磷酸结合位点，与腺嘌呤结合位点的方向大约相反180度。该研究证实ANPP是一种真正的磷酸类似物，并表明它特异性靶向CF(1)的α和β亚基上核苷酸结合口袋内的γ-磷酸结合位点。该研究还表明，在叶绿体F(1)-F(0)复合体的静止状态下，α和β亚基在结构上都是不对称的。
