Avaeva S M, Rodina E V, Vorobyeva N N, Kurilova S A, Nazarova T I, Sklyankina V A, Oganessyan V Y, Samygina V R, Harutyunyan E H
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119899, Russia.
Biochemistry (Mosc). 1998 Jun;63(6):671-84.
The three-dimensional structures of four mutant E. coli inorganic pyrophosphatases (PPases) with single Asp-->Asn substitutions at positions 42, 65, 70, and 97 were solved at 1.95, 2.15, 2.10, and 2.20 A resolution, respectively. Asp-42-->Asn and Asp-65-->Asn mutant PPases were prepared as complexes with sulfate--a structural analog of phosphate, the product of enzymatic reaction. A comparison of mutant enzymes with native PPases revealed that a single amino acid substitution changes the position of the mutated residue as well as the positions of several functional groups and some parts of a polypeptide chain. These changes are responsible for the fact that mutant PPases differ from the native ones in their catalytic properties. The sulfate binding to the mutant PPase active site causes molecular asymmetry, as shown for the native PPase earlier. The subunit asymmetry is manifested in different positions of sulfate and several functional groups, as well as changes in packing of hexamers in crystals and in cell parameters.
分别在1.95、2.15、2.10和2.20埃分辨率下解析了4种突变型大肠杆菌无机焦磷酸酶（PPases）的三维结构，这些突变体在42、65、70和97位发生了单个天冬氨酸（Asp）到天冬酰胺（Asn）的替换。将42位天冬氨酸突变为天冬酰胺（Asp-42→Asn）和65位天冬氨酸突变为天冬酰胺（Asp-65→Asn）的突变型PPases制备成与硫酸盐（酶促反应产物磷酸盐的结构类似物）的复合物。突变酶与天然PPases的比较表明，单个氨基酸替换会改变突变残基的位置以及几个官能团和多肽链某些部分的位置。这些变化导致突变型PPases在催化特性上与天然PPases不同。如之前对天然PPase的研究所示，硫酸盐与突变型PPase活性位点的结合会导致分子不对称。亚基不对称表现为硫酸盐和几个官能团处于不同位置，以及晶体中六聚体堆积和晶胞参数的变化。
