Sulfate Adenylation-Biochemistry & Enzymology

描述（由申请人提供）：硫的代谢呈现出一个复杂而迷人的分子网络，其活动影响许多生物学领域，包括人类疾病。我们现在知道，至少有四种参与细菌硫同化的酶组织成一个多功能复合物，从中产生新的催化功能（ATP水解）。值得注意的是，通过构象变化，这种水解与半胱氨酸生物合成途径中第一种酶- ATP硫化酶的周转在动力学和能量上联系在一起。我们打算探索这种联系的机制，并在体外和活细胞环境中确定半胱氨酸代谢组的组成和组织。我们发现，在III型硫酸盐活化复合物（SACs）中，活化硫酸盐（APS）在产生和消耗它的活性位点之间沿着一个深75 e长的沟槽移动，该沟槽根据APS的位置打开和关闭。使用FRET，我们将测试在APS传输过程中通道关闭形成管状结构的假设。结合预稳态和FRET测量，我们将构建一个时间轴，将催化循环中发生的事件与通道长度的距离变化相结合。利用布朗动力学，我们将提出一个前沿模型，说明这个非凡的分子机器的形状和静电的变化是如何与其中APS的运动相耦合的。硫酰部分（SO3）从活化硫酸盐到生物受体的转移被细胞广泛用于调节代谢。催化这些转移的硫转移酶受到变构底物抑制，这一点尚未得到很好的理解，主要是因为广泛的结构和功能研究尚未确定变构结合袋。人雌激素硫转移酶（EST）表现出一个预稳态产物爆发，对应于二聚体中一半的活性位点。如果EST是一种半位点活性酶，那么非催化活性位点很可能是抑制变构的位点。我们将使用人类EST来验证这一假设，EST是一种酶，其活性与乳腺癌和子宫内膜的癌症密切相关。硫酰基（SO3）从活化硫酸盐转移到各种代谢受体被细胞广泛用于调节功能。催化这些反应的硫转移酶本身受变构底物抑制的调节，尽管有相当大的努力，但其分子机制尚不清楚。我们相信我们现在了解了这一机制，并将在即将到来的资助期内证明我们的机制假设。这些酶的作用与许多人类疾病密切相关，包括：血友病B、免疫系统受损、雌雄同体、乳房和子宫内膜肿瘤。在第二篇文章中，我们将探讨结核分枝杆菌中发现的一种罕见的复合体。我们发现，这种复合物在哺乳动物中并不存在，因此有望对物种进行特异性抑制。该半胱氨酸代谢组由半胱氨酸生物合成途径中六种酶中的至少四种酶组成，并表现出显著的催化协同作用。我们将详细描述这个复合体，这是第一次。

DESCRIPTION (provided by applicant): The metabolism of sulfur presents a complex and fascinating molecular network whose activities impact many areas of biology, including human disease. We now know that at least four of the enzymes involved in sulfur assimilation in bacteria organize into a multifunctional complex from which new catalytic function (ATP hydrolysis) emerges. Remarkably, this hydrolysis is kinetically and energetically linked, via conformational changes, to turnover of the first enzyme in the cysteine biosynthetic pathway - ATP sulfurylase. We intend to explore the mechanism of this linkage and to determine the composition and organization of the cysteine-metabolome both in vitro and in the environment of a living cell. We have discovered that in Type III sulfate activating complexes (SACs), activated sulfate (APS) travels between the active sites that produce and consume it along a deep 75 E-long groove that opens and closes in response to the position of APS. Using FRET, we will test the hypothesis that the channel closes to form a tubular structure during APS transit. Combining pre-steady state and FRET measurements, we will construct a timeline that interdigitates the events that occur in the catalytic cycle with changes in distance along the length of the channel. Using Brownian Dynamics we will advance a cutting-edge model of how changes in the shape and electrostatics of this remarkable molecular machine are coupled to the movement of the APS within it. Transfer of the sulfuryl- moiety (SO3) from activated sulfate to biological recipients is used widely by the cell to regulate metabolism. Sulfotransferases, which catalyze these transfers, are subject to allosteric substrate inhibition that is not well understood primarily because extensive structural and function work has not identified an allosteric binding pocket. The human estrogen sulfotransferase (EST) exhibits a presteady-state product burst that corresponds to precisely one-half of the active sites in the dimer. If EST is a half-site reactive enzyme, the non-catalytic active site might well function as the allosteric site of inhibition. We will test this hypothesis using the human EST, an enzyme whose activity is tightly and causally linked to cancer in the breast and endometrium. PUBLIC HEALTH RELEVANCE Transfer of the sulfuryl-group (SO3) from activated sulfate to various metabolic recipients is used widely by the cell to regulate function. Sulfotransferases, which catalyze these reactions, are themselves regulated by allosteric substrate inhibition, the molecular mechanism of which is unknown despite considerable effort to the contrary. We believe we now understand this mechanism, and will prove our mechanistic hypotheses in the upcoming grant period. The actions of these enzymes are tightly, causally linked to numerous human disease conditions, including: hemophilia B, compromised immune systems, androgyny, and breast and endometrial tumors. In a second Aim, we will explore a rare complex found in M. tuberculosis. This complex, which we discovered, is not present in mammals and therefore holds the promise of species-specific inhibition. This cysteine metabolome is comprise of at least four of the six enzymes in the cysteine biosynthetic pathway, and exhibits remarkable catalytic synergies. We will characterize this complex in detail, and for the first time.