ENZYMES AND INHIBITORS

描述：肉豆蔻酰辅酶A转移酶（NMT）转移肉豆蔻酸 与靶蛋白或肽的N末端甘氨酸连接。 豆蔻酰化可能 用于将肽或蛋白质锚在疏水区域中。 机制 将研究NMT的作用。 一个重要的问题是， 肉豆蔻酸酯与酶形成共价中间体。 回答这个 问题[3 H]肉豆蔻酰CoA将在不存在肽的情况下加入NMT中 受体，或在未豆蔻酰化的肽类似物存在下。 该酶将被胰蛋白酶和胰凝乳蛋白酶降解。 所得 将肽分离并检查放射活性。 如果放射性标记 物质与其中一种肽缔合，形成共价键， 可能发生酶-底物复合物。 Aza肽是其中S1氨基酸被替换为 NH-NR-CONH。 这些肽抑制丝氨酸和巯基蛋白酶。 抑制 是由于活性位点-OH或-SH的酰化。 所得 酰基酶水解缓慢。 失活酶的结构将是 通过NMR和化学方法测定。 理解机理 对改善这些抑制剂很重要。 氮杂肽抑制剂是 对于药理学应用是有吸引力的，因为它们不受 蛋白水解消化，并且对化学降解有抗性。 他们还 是高度选择性的。 CO的形成发生在从真菌到哺乳动物的生物体中。 CO具有 它的生物学作用类似于NO，被称为神经递质。 关于CO形成的酶促机制知之甚少。 的 研究人员和他同事从K.肺炎 为了确定酶的作用机制， 将研究酶模型系统。 例如： CH 3-CO-CH 2-CO-CH 3在非酶促条件下转化为CO和其他产物， H2 O2的存在。 将进行ESR实验以检测自由基。 预计对该机制的了解将有助于 申请人设计了一种可用于抑制CO产生的抑制剂 在完整的细菌细胞中。

DESCRIPTION: The enzyme Myristoyl CoA-transferase (NMT) transfers myristate to an N terminal glycine of a target protein or peptide. Myristoylation may serve to anchor peptides or proteins in a hydrophobic region. The mechanism of action of NMT will be investigated. An important question is whether myristate forms a covalent intermediate with the enzyme. To answer this question [3H] myristoyl CoA will be added to NMT in the absence of peptide acceptor, or in the presence of a peptide analog which is not myristoylated. The enzyme will be degraded with trypsin and chymotrypsin. The resulting peptides will be separated and examined for radio-activity. If radiolabeled material is associated with one of the peptides, formation of a covalent enzyme-substrate complex probably occurred. Aza peptides are peptides in which the S1 amino acid is replaced by NH-NR-CONH. These peptides inhibit serine and thiol proteases. Inhibition is due to acylation of the active site -OH or -SH. The resulting acyl-enzyme hydrolyses slowly. The structure of the inactive enzyme will be determined by NMR and chemical approaches. Understanding of the mechanism will be important in improving these inhibitors. Aza-peptide inhibitors are attractive for pharmacological applications, since they are not subject to proteolytic digestion and are resistant to chemical degradation. They also are highly selective. CO formation occurs in organisms ranging from fungi to mammals. CO has biological effects similar to NO. It has been called a neuro transmitter. Little is known about the enzymatic mechanism of CO formation. The investigator and his colleagues have purified an enzyme from K. Pneumoniae which catalyzes the formation of CO. To ascertain the enzyme mechanism, non enzymatic model systems will be investigated. For instance: CH3-CO-CH2-CO-CH3 is non-enzymatically converted to CO and other products in the presence of H2O2. ESR experiments will be done to detect free radicals. It is anticipated that an understanding of the mechanism will enable the applicant to design an inhibitor which can be used to inhibit CO production in intact bacterial cells.