The Research Of Restructuring Arginase Activity

Arginase is a key enzyme in the process of urea cycle, which can catalyze the hydrolysis of arginine. In recent years, there is a new strategy for cancer treatment, namely, amino acid deprivation. Arginine is a non essential amino acid for normal cells, but it is an essential amino acid for cancer cells. Therefore, arginase can block tumor cells to obtain the necessary nutrition through consumption of arginine. Thereby hindering the growth of cancer cells. At the same time,there was almost no effect on normal cells. So arginase has a very good application value.Therefore more and more people begin to pay attention to the research of arginase.In the past, the research on the arginase was mainly focused on the study of its inhibitor,while the study of activator is less. Through the mechanism of amin acid deprivation to inhibit the tumor cells, we should focus on the study of its activator. Previous studies focused on the study of arginase Ⅰ. Studies on the arginase Ⅰ have also focused on the combination of the active pocket and other substances. In this paper, we focus on the study of arginase activator and arginase Ⅰ decorated by PEG. At the same time, we found that maybe there is a regulatory site in the hole C2 of arginase Ⅰ.1. Study on W-arginase Ⅰ and R-arginase Ⅰ.Through molecular dynamics simulation and PCR technique, the site directed mutagenesis of W-arginase Ⅰ was successfully obtained. Compared to W-arginase Ⅰ, the volume of C2 hole in R-arginase Ⅰ is obviously decreased, but the conformation of active pocket and the enzyme have no obvious interference. According to the molecular simulation and experimental results, it is suggested that there maybe an active site in the C2 hole, and small molecules can bind in this site, then regulate the activity of the enzyme.DMSO, glycine, ferulic acid and monosodium glutamate have activation effect on W-ArginaseⅠand R-arginaseⅠ. DMSO was the most significant for the activation of W-ArginaseⅠand R-ArginaseⅠ. DMSO, glycine, ferulic acid and monosodium glutamate have greater activation effect on R-ArginaseⅠthan W-ArginaseⅠ.2. Study on W-Arginase Ⅰ decorated by PEG.The suitable conditions for PEG modified W-Arginase Ⅰ were as follow: the temperaturewas 4 ℃, the ratio of PEG to W-Arginase Ⅰ was 16:1, and the modified time was 1h. The conditions for the separation of PEG modified and unmodified protein were by gradient elution,the low concentration side is p H6.0 of 20 m M PBS without Na Cl, the high concentration side is p H6.0 of 20 m M PBS with 200 m M Na Cl. DMSO, glycine, ferulic acid and monosodium glutamate have activation effect on W-Arginase Ⅰ decorated by PEG. DMSO, glycine, ferulic acid and monosodium glutamate have greater activation effect on W-Arginase Ⅰ than W-Arginase Ⅰ decorated by PEG.In this study, we designed a R-Arginase Ⅰ, we found that the surface of the W-Arginase Ⅰmay have another new regulatory site, which is different from the active pocket, we study on the conditions for the PEG modified W-Arginase Ⅰ and the following separation, at the same time,we study the activation effect by DMSO, glycine, ferulic acid and monosodium glutamate on W-Arginase Ⅰ, R-Arginase Ⅰ and W-Arginase Ⅰ decorated by PEG. This study is expected to provide a new way of thinking for research of drug by Arginase target.