Disruption of the aldolase A tetramer into catalytically active monomers
Disruption of the aldolase A tetramer into catalytically active monomers
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DOI：
10.1073/pnas.93.11.5374
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发表时间：
1996-05-28
影响因子：
11.1
通讯作者：
Tolan, DR
中科院分区：
文献类型：
--
作者：
Beernink, PT;Tolan, DR
果糖-1，6-二磷酸醛缩酶（EC 4.1.2.13）同源四聚体已经通过在两个不同亚基界面处的定点诱变而不稳定。一个双突变型醛缩酶，Q125 D/E224 A，沉淀为两个不同的物种，一个缓慢的平衡的特点，与预期的单体和四聚体的速度。醛缩酶单体被证明是催化活性的分离后，从蔗糖密度梯度。分离的醛缩酶单体具有野生型酶的比活性的72%和略低的米氏常数，清楚地表明四级结构不是催化所需的。分离的单体的交联证实了其在分离后不会与四聚体快速再平衡。四聚体和单体在被尿素灭活方面存在实质性差异。这些寡聚体变体对尿素和热失活的稳定性表明四级结构在维持醛缩酶稳定性中的作用，这可能是四级结构在许多蛋白质中的重要作用。
The fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) homotetramer has been destabilized by site-directed mutagenesis at the two different subunit interfaces. A double mutant aldolase, Q125D/E224A, sediments as two distinct species, characteristic of a slow equilibrium, with velocities expected for the monomer and tetramer. The aldolase monomer is shown to be catalytically active following isolation from sucrose density gradients. The isolated aldolase monomer had 72% of the specific activity of the wild-type enzyme and a slightly lower Michaelis constant, clearly indicating that the quaternary structure is not required for catalysis. Cross-linking of the isolated monomer confirmed that it does not rapidly reequilibrate with the tetramer following isolation. There was a substantial difference between the tetramer and monomer in their inactivation by urea. The stability toward both urea and thermal inactivation of these oligomeric variants suggests a role for the quaternary structure in maintaining the stability of aldolase, which may be an important role of quaternary structure in many proteins.