Matsui T, Jault J M, Allison W S, Yoshida M
Research Laboratory of Resource Utilization, R-1, Tokyo Institute of Technology, Yokohama 226, Japan.
Biochem Biophys Res Commun. 1996 Mar 7;220(1):94-7. doi: 10.1006/bbrc.1996.0362.
The alpha and beta subunits of F1-ATPase are homologous in primary structure and have similar folding topologies. The position of the essential Glu residue in the catalytic sites which reside in the beta subunits is occupied by a Gln residue in the noncatalytic nucleotide binding sites which reside in the alpha subunits. To test if an exchange of catalytic and noncatalytic binding sites is possible, we have replaced the Gln-Lys sequence in the noncatalytic binding site of the alpha subunit with Glu-Arg and, reciprocally, the Glu in the catalytic site of the beta subunit with Gln. The resultant mutant alpha3beta3gamma complex lost steady-state ATPase activity. However, HPLC analysis of tryptic digests of the mutant alpha3beta3gamma complex which had been photolabeled with 2-N3-[8-3H]ATP revealed that ATP tethered to the noncatalytic binding side was hydrolyzed, indicating that a primitive catalytic ability was generated at the alpha subunit by the introduced Glu.
F1 - ATP酶的α亚基和β亚基在一级结构上具有同源性，并且具有相似的折叠拓扑结构。位于β亚基催化位点的必需谷氨酸残基的位置，在位于α亚基的非催化核苷酸结合位点中被谷氨酰胺残基占据。为了测试催化和非催化结合位点的交换是否可行，我们已将α亚基非催化结合位点中的谷氨酰胺 - 赖氨酸序列替换为谷氨酸 - 精氨酸，反之，将β亚基催化位点中的谷氨酸替换为谷氨酰胺。所得的突变体α3β3γ复合物失去了稳态ATP酶活性。然而，对用2 - N3 - [8 - 3H]ATP进行光标记的突变体α3β3γ复合物的胰蛋白酶消化产物进行HPLC分析表明，与非催化结合侧相连的ATP被水解，这表明通过引入的谷氨酸在α亚基上产生了原始的催化能力。
