Sulfate Adenylation-Biochemistry & Enzymology

描述(申请人提供)：硫的新陈代谢呈现了一个复杂而迷人的分子网络，其活动影响了生物学的许多领域，包括人类疾病。我们现在知道，细菌中至少有四种参与硫同化的酶组织成一个多功能复合体，从中出现新的催化功能(ATP水解)。值得注意的是，这种水解通过构象变化与半胱氨酸生物合成途径中的第一个酶--三磷酸腺苷硫酰基酶的周转密切相关。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体的组成和组织。我们发现，在III型硫酸盐活化复合体(SACS)中，活化的硫酸盐(APS)沿着一条75E长的深沟在生产和消费它的活性中心之间移动，该凹槽根据APS的位置而打开和关闭。使用FRET，我们将检验通道在APS转运过程中关闭以形成管状结构的假设。结合稳态前测量和FRET测量，我们将构建一条时间线，将催化循环中发生的事件与沿通道长度的距离变化交错。利用布朗动力学，我们将提出一个尖端模型，说明这台非凡的分子机器的形状和静电学的变化是如何与其内部APS的运动相耦合的。硫酰基团(SO3)从活化的硫酸盐转移到生物受体被细胞广泛用于调节新陈代谢。催化这些转移的磺基转移酶受到变构底物的抑制，这主要是因为广泛的结构和功能研究尚未确定变构结合口袋。人类雌激素磺基转移酶(EST)表现出一个稳定前的产物爆发，恰好对应于二聚体中一半的活性部位。如果EST是半活性部位的酶，那么非催化活性部位很可能是抑制的变构部位。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癌密切相关。硫酰基(SO3)从活化的硫酸盐转移到各种代谢受体的公共健康相关性转移被细胞广泛用于调节功能。催化这些反应的磺基转移酶本身受变构底物抑制的调节，尽管有相当多的相反的努力，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，并将在即将到来的赠款期间证明我们的机械论假说。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、雌雄同体以及乳腺和子宫内膜肿瘤。在第二个目标中，我们将探索在结核分枝杆菌中发现的一种罕见的复合体。我们发现，这种复合体在哺乳动物中不存在，因此具有物种特异性抑制的前景。这个半胱氨酸代谢体由半胱氨酸生物合成途径中的六种酶中的至少四种组成，并显示出显著的催化协同作用。我们将首次详细描述这一综合体的特征。

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.