HIV 1 PROTEASE CALORIMETRY: NON CHROMOGENIC SUBSTRATE FOR ENZYMATIC ACTIVITY

HIV 1 蛋白酶量热法：酶活性的非显色底物

• 批准号: 6122051
• 负责人: MATTHEW J TODD
• 金额: --
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-05 至 1998-08-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6122051
• 关键词: AIDS; biological products; biomaterials; biomedical resource; communicable diseases; education; human tissue; immunology; lymphatic system; model design /development; structural biology; technology /technique development; virus

A novel universal method for measuring enzymatic activity using micro-calorimetry is applied to the HIV-1 protease (PR). Biocalorimetric assays measure the rate of heat evolution during conversion of substrate to product. Calorimetry thus gives a direct measure of the rate of an enzymatic reaction, as opposed to conventional assays where rate is calculated as a derivative of a quantity with time. Since no chromophore is necessary, the physiological substrates can be used. The activity of PR mutant (Q7K, L33I, L63I) was assayed using: 1) a standard chromogenic substrate (Arg-Ala-Arg-Val-Nle-p-nitro-Phe-Glu-Ala-Nle-NH2); 2) a non-chromogenic version of the same peptide (Arg-Ala-Arg-Val-Nle-p-nitro-Phe-Glu-Ala-Nle-NH2); and, 3) a non-chromogenic physiological substrate (Ser-Gln-Asn-Tyr-Pro-Ile-Val-NH2). The activity of PR as a function of salt concentration, pH, temperature (and <Cp>) for Km and Vmax are described. In addition, the inhibition by products pepstatin and acetyl-pepstain is described.

一种新的通用酶活性测定方法 微量热法应用于HIV-1蛋白酶（PR）。 生物量热测定测量过程中的放热速率 底物转化为产物。 因此，量热法给出了直接的 酶促反应速率的量度，与 其中速率被计算为 数量与时间 由于不需要发色团， 可以使用生理底物。 PR突变体（Q7 K， L33 I，L 63 I）使用以下物质测定：1）标准显色底物 （Arg-Ala-Arg-Val-Nle-p-nitro-Phe-Glu-Ala-Nle-NH2）; 相同肽的非显色形式 （Arg-Ala-Arg-Val-Nle-p-硝基-Phe-Glu-Ala-Nle-NH 2）;和 非显色生理底物 （Ser-Gln-Asn-Tyr-Pro-Ile-Val-NH2）。 PR作为一种功能 Km和Vmax的盐浓度、pH、温度（和<Cp>）为 介绍了 此外该 描述了抑制副产物胃蛋白酶抑制剂和乙酰胃蛋白酶抑制剂。