The mechanism of velocity modulated allosteric regulation in D-3-phosphoglycerate dehydrogenase - Cross-linking adjacent regulatory domains with engineered disulfides mimics effector binding
The mechanism of velocity modulated allosteric regulation in D-3-phosphoglycerate dehydrogenase - Cross-linking adjacent regulatory domains with engineered disulfides mimics effector binding
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DOI：
10.1074/jbc.271.22.13013
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发表时间：
1996-05-31
影响因子：
4.8
通讯作者：
Grant, GA
中科院分区：
文献类型：
--
作者：
AlRabiee, R;Lee, EJ;Grant, GA
来自大肠杆菌的D-3-磷酸甘油酸脱氢酶（PGDH）（EC 1.1.1.95）是V-max型的变构调节酶，它是相同亚基的四聚体，并且每个亚基由三个可识别的结构域组成，辅因子结合结构域、底物结合结构域和调节结构域，每个亚基通过相邻的辅因子结合结构域和通过相邻的调节结构域、L-丝氨酸（生理效应物）、通过与每个亚基形成氢键将相邻亚基的调节结构域明显地拴在一起来抑制催化活性。该研究表明，在不存在抑制剂的情况下，用工程化二硫化物交联相邻的调节结构域以与抑制剂产生的方式类似的方式产生催化抑制，由交联引起的抑制可以通过二硫苏糖醇以浓度依赖性方式完全逆转。含有还原状态的工程化半胱氨酸的活性突变酶保留其被L-丝氨酸抑制的能力，尽管在100倍高的浓度下，突变体和天然酶的丝氨酸抑制的Hill图表明，在突变体酶中相互作用位点的数目保持在2，用工程化的二硫键束缚相邻的调节结构域导致酶活性的可逆抑制，这表明这些结构域以某种方式相对移动，这些观察结果支持该模型，该模型预测催化活性受刚性结构域围绕柔性铰链的运动调节，并且效应物结合通过将调节结构域锁定在产生开放活性位点裂缝的状态来防止这种情况。
D-3-Phosphoglycerate dehydrogenase (PGDH) (EC 1.1.1.95) from Escherichia coli is an allosterically regulated enzyme of the V-max type, It is a tetramer of identical subunits and each subunit is made up of three identifiable domains, the cofactor binding domain, the substrate binding domain, and the regulatory domain, Each subunit contacts two other subunits through adjacent cofactor binding domains and through adjacent regulatory domains, L-Serine, the physiological effector, inhibits catalytic activity by apparently tethering regulatory domains from adjacent subunits together through the formation of hydrogen bonds to each subunit, This investigation demonstrates that cross-linking adjacent regulatory domains with engineered disulfides produces catalytic inhibition in the absence of inhibitor in a manner similar to that produced by the inhibitor, The inhibition due to cross-linking can be completely reversed in a concentration dependent manner by dithiothreitol, The active mutant enzyme, containing the engineered cysteines in the reduced state, retains its ability to be inhibited by L-serine, although at a 100-fold higher concentration, Hill plots of the serine inhibition of mutant and native enzyme indicate that the number of interacting sites remains at 2 in the mutant enzyme, The reversible inhibition of enzyme activity that results from tethering adjacent regulatory domains with engineered disulfides suggests that these domains move in some manner relative to one another during the active to inhibited state transition, These observations support the model which predicts that catalytic activity is regulated by the movement of rigid domains about flexible hinges and that effector binding prevents this by locking the regulatory domains in a state that produces an open active site cleft.