Stability and Mechanism of Action of the Catalytic Subunit of Aspartate Transcarbamoylase

从大肠杆菌天冬氨酸氨基转移酶(ATCase)中分离出的高活性催化(C)亚基与调节亚基相互作用形成完整的ATCase分子，该分子由6条催化和6条调节多肽链组成，C亚基发生显著变化，导致酶活性急剧下降。此外，随着底物的加入，组装的复合体中的C亚基发生了进一步的广泛变化，导致了更活跃的酶。是什么结构和催化特征赋予C三聚体这些特殊的性质，从而导致C三聚体与大多数寡聚酶显着不同？了解结构的稳定性和可能的波动将是无价的。连接N-末端和C-末端结构域的长螺旋起什么作用，每个结构域约由140个氨基酸组成？在需要来自相邻链的氨基酸残基的共同参与的C三聚体中，共享活性中心的意义是什么？链间盐键在稳定C三聚体方面起什么作用？不同结构域的作用是什么？其催化机理是什么？要回答这些问题，需要通过定点突变构建许多不同的突变形式，确定对催化活性和链的正确折叠至关重要的特定氨基酸的作用，使用核磁共振光谱测量与催化有关的残基的pk，分析广泛pH范围内的活性模式以评估酸碱催化，研究同位素效应以阐明催化机制，以及研究过渡态类似物抑制剂的结合。

When highly active catalytic (C) subunits isolated from Escherichia coli aspartate transcarbamoylase (ATCase) interact with regulatory subunits to form intact ATCase molecules, composed of 6 catalytic and 6 regulatory polypeptide chains, there are striking changes in the C subunits leading to a dramatic decrease in enzyme activity. Moreover, C subunits in the assembled complex undergo further extensive changes upon the addition of substrates, leading a more active enzyme. What are the structural and catalytic features which endow C trimers with these special properties thereby causing the C trimer to differ markedly from most oligomeric enzymes? Knowledge of the stability and possible fluctuations in structure would be invaluable. What is the role of the long helix connecting the N- terminal and C-terminal domains, each composed about 140 amino acids? What is the significance of shared active sites in the C trimer requiring joint participation of amino acid residues from adjacent chains? What role do interchain salt-links play in stabilizing C trimers and what is the role of distinct domains? What is the catalytic mechanism? Answering these questions requires many different mutant forms constructed by site-directed mutagenesis, determining the role of specific amino acids crucial for catalytic activity and correct folding of the chains, using NMR spectroscopy to measure pK's of residues implicated in catalysis, analyzing the pattern of activity over a broad pH range to evaluate acid-base catalysis, studying isotope effects to elucidate the catalytic mechanism, and studying binding of transition state analog inhibitors.