Pagan J, Senior A E
Department of Biochemistry, University of Rochester Medical Center, NY 14642.
FEBS Lett. 1990 Oct 29;273(1-2):147-9. doi: 10.1016/0014-5793(90)81071-u.
It is shown that ATP dissociates very slowly (koff less than 6.4 x 10(5) s-1, t1/2 greater than 3 h) from the three noncatalytic sites of E. coli F1-ATPase and that ADP dissociates from these three sites in a homogeneous fashion with koff = 1.5 x 10(-4) s-1 (t1/2 = 1.35 h). Mutagenesis of alpha-subunit residues R171 and Q172 in the 'glycine-rich loop' (Homology A) consensus region of the noncatalytic sites was carried out to test the hypothesis that unusually bulky residues at these positions are responsible wholly or partly for the observed tight binding of adenine nucleotides. The mutations alpha Q172G or alpha R171S,Q172G had no effects on ATP or ADP binding to or rates of dissociation from F1 noncatalytic sites. KdATP and KdADP of isolated alpha-subunit were weakened by approximately 1 order of magnitude in both mutants. The results suggest that neither residue alpha R171 nor alpha Q172 interacts directly with bound nucleotide, and show that the presence of bulky residues per se in the glycine-rich loop region of F1-alpha-subunit is not responsible for tight binding in the noncatalytic sites.
结果表明，ATP从大肠杆菌F1 - ATP酶的三个非催化位点解离非常缓慢（解离常数koff小于6.4×10⁵ s⁻¹，半衰期t1/2大于3小时），而ADP以均匀的方式从这三个位点解离，解离常数koff = 1.5×10⁻⁴ s⁻¹（半衰期t1/2 = 1.35小时）。对非催化位点“富含甘氨酸环”（同源性A）保守区域中的α亚基残基R171和Q172进行诱变，以检验以下假设：这些位置异常庞大的残基全部或部分导致了观察到的腺嘌呤核苷酸紧密结合。αQ172G或αR171S,Q172G突变对ATP或ADP与F1非催化位点的结合或解离速率没有影响。在两个突变体中，分离的α亚基的KdATP和KdADP减弱了约1个数量级。结果表明，αR171和αQ172残基均不直接与结合的核苷酸相互作用，并表明F1 - α亚基富含甘氨酸环区域中本身存在庞大残基并非非催化位点紧密结合的原因。
