Sulfate Adenylation-Biochemistry & Enzymology

描述(由申请人提供)： 硫酸盐进入新陈代谢需要被化学激活。唯一已知的活化硫酸盐的代谢方法是形成非常高能的磷酸-硫酸酸酐键(？go=-19千卡/摩尔)。该键是活性硫酸盐(APS或PAPS)的化学标志，正是从这种高能环境中，硫酰部分(-SO3)迅速和有利地进入其后续的代谢生物化学。活化键是在三磷酸腺苷硫酰转移酶催化的转移反应中形成的，在转移反应中，三磷酸腺苷(AMP~)部分转移到硫酸盐上。在哺乳动物中，硫基转移到蛋白质和小分子代谢物调节着广泛的代谢过程，包括神经肽和类固醇激素作用、生长因子识别和淋巴细胞循环。这项提案概述了旨在解决关于哺乳动物类ATP硫酰基酶的功能和进化的中心问题的基于结构的机械性研究。 细菌三磷酸腺苷硫酰基酶含有一个GTP酶亚基(在本实验室发现)，它是延伸因子Tu的进化后代。该亚基的构象变化加速了腺酰转移酶亚基的周转，并耦合了GTP水解和APS合成的化学势。我们最近发现，三磷酸腺苷硫化酶与半胱氨酸生物合成途径中的另一种酶(O-乙酰-L-丝氨酸硫化酶)形成复合体，并相互作用产生新的催化功能--三磷酸腺苷的水解。这些酶组织成一个新陈代谢泵，每一次冲程都会将一个APS分子送入代谢途径。在这笔赠款中，将对泵的机理进行探索。使用一种尚未表征的来自结核分枝杆菌的新型三磷酸腺苷硫酰酶，我们的初步数据将这些发现扩展到包括该途径中的七种酶中的五种。我们将定义和研究半胱氨酸代谢蛋白，目的是了解这一途径的自组织所产生的层级功能。

DESCRIPTION (provided by applicant): The entry of sulfate into metabolism requires that it be chemically activated. The only known metabolic means of activating sulfate is the formation of the very high-energy phosphoric-sulfuric acid anhydride bond (?Go= -19 kcal/mole). This bond is the chemical hallmark of activated sulfate (APS or PAPS), and it is from this high-energy environment that the sulfuryl-moiety (-SO3) passes quickly and favorably into its subsequent metabolic biochemistry. The activated bond is formed in a transfer reaction, catalyzed by ATP sulfurylase, in which the adenylyl-moiety (AMP~) of ATP is transferred to sulfate. In mammals, sulfuryl-group transfer to proteins and small molecule metabolites regulates a wide-variety of metabolic processes including neuropeptide- and steroid-hormone action, growth-factor recognition, and lymph cell circulation. This proposal outlines structurally-based mechanistic inquires designed to address central issues regarding the function and evolution of the mammalian class of ATP sulfurylases. Bacterial ATP sulfurylases harbor a GTPase subunit (discovered in this laboratory) that is an evolutionary descendant of elongation factor Tu. The conformational changes that this subunit undergoes as a consequence of GTP hydrolysis accelerate turnover of the adenylyl-transferase subunit, and couple the chemical potentials of GTP hydrolysis and APS synthesis. We have recently discovered that ATP sulfurylase forms a complex with another enzyme in the cysteine biosynthetic pathway (O-acetly-l-serine sulfhydrylase), and that their interactions produce "new" catalytic function - the hydrolysis of ATP. These enzymes organize into a metabolic pump, each stroke of which delivers one molecule of APS into the pathway. The mechanism of the pump will be explored in this grant. Working with an as yet uncharacterized and novel ATP sulfurylase from Mycobacterium tuberculosis, our preliminary data extends these finding to include five of the seven enzymes in the pathway. We will define and study the cysteine metabolon with the goal of understanding the hierarchical functions that emerge from the self-organization of this pathway.