Maldonado A M, Portillo F
Departamento de Bioquímica, Facultad de Medicina, Universidad Autónoma de Madrid, Spain.
J Biol Chem. 1995 Apr 14;270(15):8655-9. doi: 10.1074/jbc.270.15.8655.
The highly conserved motif of Saccharomyces cerevisiae H(+)-ATPase 474KGAP has been proposed to participate in the formation of the phosphorylated intermediate during the catalytic cycle (Portillo, F., and Serrano, R. (1988) EMBO J. 7, 1793-1798). In addition, Lys-474 is the FITC binding site of the yeast enzyme (Portillo, F. and Serrano, R. (1989) Eur. J. Biochem. 186, 501-507). We have performed an intragenic suppressor analysis of the K474R mutation to identify the interacting regions involved in these functions. Random in vitro mutagenesis of the K474R allele resulted in seven suppressor (second-site) mutations. One mutation (V396I), located 18 residues away from the Asp-378 residue, which is phosphorylated during catalysis, is allele-specific. This provides genetic evidence of a direct interaction between the KGAP motif and the phosphorylation domain during the catalytic cycle. Three mutations (V484I, V484I/E485K, and E485K/E486K) are located near Lys-474 and may compense the structural alteration introduced by the K474R mutation. Two substitutions at the end of the predicted transmembrane stretch 2 (A165V and V169I/D170N) and another in the predicted ATP binding domain (P536L) may act as allele-nonspecific suppressors, as they are also able to suppress a mutation at the enzyme's carboxyl terminus.
酿酒酵母H(+)-ATP酶474KGAP的高度保守基序被认为在催化循环中参与磷酸化中间体的形成（波蒂略，F.，和塞拉诺，R.（1988年）《欧洲分子生物学组织杂志》7，1793 - 1798）。此外，赖氨酸-474是酵母酶的异硫氰酸荧光素结合位点（波蒂略，F.和塞拉诺，R.（1989年）《欧洲生物化学杂志》186，501 - 507）。我们对K474R突变进行了基因内抑制分析，以确定参与这些功能的相互作用区域。K474R等位基因的随机体外诱变产生了七个抑制（第二位点）突变。一个突变（V396I），位于距催化过程中被磷酸化的天冬氨酸-378残基18个残基处，是等位基因特异性的。这提供了催化循环中KGAP基序与磷酸化结构域之间直接相互作用的遗传学证据。三个突变（V484I、V484I/E485K和E485K/E486K）位于赖氨酸-474附近，可能补偿K474R突变引入的结构改变。预测的跨膜区段2末端的两个取代（A165V和V169I/D170N）以及预测的ATP结合结构域中的另一个取代（P536L）可能作为等位基因非特异性抑制子，因为它们也能够抑制酶羧基末端的突变。
