Schroeder Gottfried K, Wolfenden Richard
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Biochemistry. 2007 Nov 27;46(47):13638-47. doi: 10.1021/bi701480f. Epub 2007 Nov 1.
To estimate the relative importance of alternate routes of spontaneous degradation of DNA and the rate enhancements produced by enzymes catalyzing these reactions, rate constants and thermodynamic activation parameters for the degradation of 2'-deoxynucleosides at 25 degrees C were determined by extrapolation of rates observed in the temperature range between 90 and 200 degrees C in neutral phosphate buffer. Rates of deamination of 2'-deoxycytidine, 1-methylcytosine, and cytidine were found to be identical within experimental error (t1/2 approximately 20 years, 37 degrees C). Rate constants for deamination of 2'-deoxyadenosine and 2'-deoxyguanosine, which could not be determined directly because of rapid glycoside cleavage, were estimated by assuming that methyl replacement should generate reasonable model substrates. The rates of deamination of 9-methyladenine and 9-methylguanine were found to be similar to each other (t1/2 approximately 6000 years, 37 degrees C) and approximately 10(2)-fold slower than the rates of glycoside cleavage in 2'-deoxyadenosine and 2'-deoxyguanosine. The deamination of 2'-deoxyadenosine, 2'-deoxyguanosine, and 2'-deoxycytidine led to accelerated rates of glycoside cleavage. In the exceptional case of 2'-deoxycytidine, deamination and glycoside hydrolysis proceed at very similar rates at all temperatures. Glycoside cleavage proceeds with half-times ranging from 4 years for 2'-deoxyinosine to 40 years for 2'-deoxycytidine (37 degrees C). The rate enhancements produced by DNA glycosylases, estimated by comparison with the rates of these uncatalyzed reactions, are found to be substantially smaller than those produced by deaminases and staphylococcal nuclease.
为了评估DNA自发降解的替代途径的相对重要性以及催化这些反应的酶所产生的速率增强作用，通过外推在90至200摄氏度的温度范围内于中性磷酸盐缓冲液中观察到的速率，测定了25摄氏度时2'-脱氧核苷降解的速率常数和热力学活化参数。发现在实验误差范围内，2'-脱氧胞苷、1-甲基胞嘧啶和胞苷的脱氨速率是相同的（半衰期约为20年，37摄氏度）。由于糖苷快速裂解而无法直接测定2'-脱氧腺苷和2'-脱氧鸟苷的脱氨速率常数，通过假设甲基取代应产生合理的模型底物来进行估算。发现9-甲基腺嘌呤和9-甲基鸟嘌呤的脱氨速率彼此相似（半衰期约为6000年，37摄氏度），并且比2'-脱氧腺苷和2'-脱氧鸟苷中的糖苷裂解速率慢约10²倍。2'-脱氧腺苷、2'-脱氧鸟苷和2'-脱氧胞苷的脱氨导致糖苷裂解速率加快。在2'-脱氧胞苷这种特殊情况下，脱氨和糖苷水解在所有温度下的进行速率都非常相似。糖苷裂解的半衰期从2'-脱氧次黄苷的4年到2'-脱氧胞苷的40年不等（37摄氏度）。通过与这些无催化反应的速率进行比较估算得出，DNA糖基化酶所产生的速率增强作用明显小于脱氨酶和葡萄球菌核酸酶所产生的速率增强作用。
