目的:对重组尿酸酶和聚乙二醇化重组尿酸酶的酶动力学特征进行研究。 方法:建立底物消耗法和产物生成法2种重组尿酸酶活性的测定方法,研究表明二者测定酶活性结果一致。选择尿酸底物消耗法对聚乙二醇修饰前后重组尿酸酶的活性及酶动力学参数进行比较分析。 结果:重组尿酸酶在37 ℃、最适pH 9.0时,对应的Vmax为0.013 μmol·L-1·min-1、Km为33.77 μmol·L-1、Kcat为889.0 min-1;聚乙二醇修饰重组尿酸酶在37 ℃、最适pH 9.5时,对应的Vmax为0.009 μmol·L-1·min-1、Km为31.52 μmol·L-1、Kcat为615.5 min-1。在相应最适pH条件下,聚乙二醇修饰前后重组尿酸酶的酶比活性均为12.5 U·mg-1。 结论:在最适pH条件下,重组尿酸酶经聚乙二醇修饰前后酶比活性和Km值一致,说明聚乙二醇修饰未影响重组尿酸酶的酶比活性,且聚乙二醇修饰前后酶与底物的亲和力未发生改变。与重组尿酸酶比较,聚乙二醇化重组尿酸酶的Vmax降低了约30%,说明聚乙二醇修饰可能会导致尿酸酶的四聚体构象发生改变,从而影响最大反应速度。
Objective: To study the enzyme kinetic characteristics of recombinant Uricase and PEGylated Uricase. Methods: Two methods for the determination of recombinant uricase activity, substrate depletion method and product generation method were established; the results from both assays were consistent. The urine substrate depletion method was chosen to compare the activity and enzyme kinetic parameters of uricase with or without polyglycol modification. Results: Within the optimal pH (9.0) at 37 ℃, the related Vmax, Km and Kcat of free recombinant Uricase were 0.013 μmol·L-1·min-1, 33.77 μmol·L-1 and 889.0 min-1, respectively; while the optimal pH for recombinant Uricase with polyglycol modification was 9.5 at 37 ℃ with Vmax 0.009 μmol·L-1·min-1, Km 31.52 μmol·L-1 and Kcat 615.5 min-1 correspondingly. Under the optimum pH conditions, the specific enzyme activity of Uricase with and without PEG modification,were both constant at 12.5 U·mg-1. Conclusions: Under the optimum pH conditions, the enzyme activity as well as the Km value of the recombinant uricase with or without PEG modification were consistent, indicating that the ratio activity of the recombinant Uricase was not affected by PEG modification, niether the affinity to substrates. Moreover, compared with the free recombinant Uricase, the Vmax of PEG modified enzyme shows about 30% decrease, indicating that the PEG modification may cause the change of tetramer conformation, which could affect the maximum reaction rate of Uricase.
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