Tibbitts T T, Xu X, Kantrowitz E R
Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167-3860.
Protein Sci. 1994 Nov;3(11):2005-14. doi: 10.1002/pro.5560031113.
Using site-directed mutagenesis, an aspartate side chain involved in binding metal ions in the active site of Escherichia coli alkaline phosphatase (Asp-369) was replaced, alternately, by asparagine (D369N) and by alanine (D369A). The purified mutant enzymes showed reduced turnover rates (kcat) and increased Michaelis constants (Km). The kcat for the D369A enzyme was 5,000-fold lower than the value for the wild-type enzyme. The D369N enzyme required Zn2+ in millimolar concentrations to become fully active; even under these conditions the kcat measured for hydrolysis of p-nitrophenol phosphate was 2 orders of magnitude lower than for the wild-type enzyme. Thus the kcat/Km ratios showed that catalysis is 50 times less efficient when the carboxylate side chain of Asp-369 is replaced by the corresponding amide; and activity is reduced to near nonenzymic levels when the carboxylate is replaced by a methyl group. The crystal structure of D369N, solved to 2.5 A resolution with an R-factor of 0.189, showed vacancies at 2 of the 3 metal binding sites. On the basis of the kinetic results and the refined X-ray coordinates, a reaction mechanism is proposed for phosphate ester hydrolysis by the D369N enzyme involving only 1 metal with the possible assistance of a histidine side chain.
利用定点诱变技术，将大肠杆菌碱性磷酸酶活性位点中参与结合金属离子的天冬氨酸侧链（Asp-369），依次替换为天冬酰胺（D369N）和丙氨酸（D369A）。纯化后的突变酶显示出周转率（kcat）降低，米氏常数（Km）增加。D369A酶的kcat值比野生型酶低5000倍。D369N酶需要毫摩尔浓度的Zn2+才能完全激活；即使在这些条件下，对磷酸对硝基苯酚水解测得的kcat值仍比野生型酶低2个数量级。因此，kcat/Km比值表明，当Asp-369的羧酸盐侧链被相应的酰胺取代时，催化效率降低50倍；当羧酸盐被甲基取代时，活性降低到接近非酶水平。D369N的晶体结构分辨率为2.5 Å，R因子为0.189，显示在3个金属结合位点中的2个有空位。基于动力学结果和精细的X射线坐标，提出了D369N酶催化磷酸酯水解的反应机制，该机制仅涉及1种金属，可能有组氨酸侧链的协助。
