Substrate-Induced Facilitated Dissociation of the Competitive Inhibitor from the Active Site of O-Acetyl Serine Sulfhydrylase Reveals a Competitive-Allostery Mechanism,Biochemistry

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Substrate-Induced Facilitated Dissociation of the Competitive Inhibitor from the Active Site of O-Acetyl Serine Sulfhydrylase Reveals a Competitive-Allostery Mechanism
Biochemistry ( IF 2.9 ) Pub Date : 2017-09-01 00:00:00 , DOI: 10.1021/acs.biochem.7b00500
Appu Kumar Singh ₁ , Mary Krishna Ekka ₁ , Abhishek Kaushik ₁ , Vaibhav Pandya ₁ , Ravi P. Singh ₁ , Shrijita Banerjee ₁ , Monica Mittal ₁ , Vijay Singh ₁ , S. Kumaran ₁

Affiliation

By classical competitive antagonism, a substrate and competitive inhibitor must bind mutually exclusively to the active site. The competitive inhibition of O-acetyl serine sulfhydrylase (OASS) by the C-terminus of serine acetyltransferase (SAT) presents a paradox, because the C-terminus of SAT binds to the active site of OASS with an affinity that is 4–6 log-fold (10⁴–10⁶) greater than that of the substrate. Therefore, we employed multiple approaches to understand how the substrate gains access to the OASS active site under physiological conditions. Single-molecule and ensemble approaches showed that the active site-bound high-affinity competitive inhibitor is actively dissociated by the substrate, which is not consistent with classical views of competitive antagonism. We employed fast-flow kinetic approaches to demonstrate that substrate-mediated dissociation of full length SAT–OASS (cysteine regulatory complex) follows a noncanonical “facilitated dissociation” mechanism. To understand the mechanism by which the substrate induces inhibitor dissociation, we resolved the crystal structures of enzyme·inhibitor·substrate ternary complexes. Crystal structures reveal a competitive allosteric binding mechanism in which the substrate intrudes into the inhibitor-bound active site and disengages the inhibitor before occupying the site vacated by the inhibitor. In summary, here we reveal a new type of competitive allosteric binding mechanism by which one of the competitive antagonists facilitates the dissociation of the other. Together, our results indicate that “competitive allostery” is the general feature of noncanonical “facilitated/accelerated dissociation” mechanisms. Further understanding of the mechanistic framework of “competitive allosteric” mechanism may allow us to design a new family of “competitive allosteric drugs/small molecules” that will have improved selectivity and specificity as compared to their competitive and allosteric counterparts.

中文翻译：

底物诱导的竞争性抑制剂从O-乙酰丝氨酸巯基水解酶活性位点的解离揭示了竞争性变构机制

通过经典的竞争拮抗作用，底物和竞争性抑制剂必须相互排他地结合到活性位点上。丝氨酸乙酰转移酶（SAT）的C末端对O-乙酰丝氨酸巯基化酶（OASS）的竞争性抑制是一个悖论，因为SAT的C末端以4–6 log的亲和力与OASS的活性位点结合倍（10 ⁴ –10 ⁶）大于基材。因此，我们采用了多种方法来了解底物在生理条件下如何获得对OASS活性位点的访问。单分子和集合方法表明，活性位点结合的高亲和力竞争性抑制剂被底物主动解离，这与经典的竞争性拮抗观点不一致。我们采用快速流动动力学方法证明全长SAT-OASS（半胱氨酸调节复合物）的底物介导的解离遵循非规范的“促进解离”机制。为了了解底物诱导抑制剂解离的机理，我们解析了酶·抑制剂·底物三元复合物的晶体结构。晶体结构揭示了竞争性的变构结合机制，其中底物侵入抑制剂结合的活性位点，并在占据抑制剂空出的位点之前脱离抑制剂。总而言之，在这里我们揭示了一种新型的竞争性变构结合机制，一种竞争性拮抗剂通过这种机制促进了另一种的解离。总之，我们的结果表明，“竞争性变构”是非规范性“促进/加速解离”机制的一般特征。对“竞争性变构”机制的机械框架的进一步理解可以使我们设计一个新的“竞争性变构药物/小分子”家族，与竞争性和变构对应物相比，具有更高的选择性和特异性。

更新日期：2017-09-04

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