本報告是利用大腸桿菌表達E3及其定點突變蛋白並用光譜分析研究此酵素之催化機制。K37E突變蛋白是在E3缺乏症的病人確認得知,另外兩個突變則是先前研究活性鹼(Active-site base H-452)及其離子對(Ion pair Glu-457)在酵素反應中扮演的角色時所創造。螢光光譜分析闡明這些胺基酸在酵素活性上的功能。H452Q突變嚴重影響此酵素之正反應(Physiological reaction);E457Q突變影響此酵素逆反應之進行;K37E突變則不對酵素反應造成影響。
In this report, the overexpression and single-step purification of recombinant wild-type and site-directed mutants of human dihydrolipoamide dehydrogenase in Escherichia coli and spectroscopic studies aimed at understanding the catalytic mechanism of this enzyme. One mutation (K37E) has been identified in a patient lacking dihydrolipoamide dehydrogenase activity, while the other two mutations were previously generated specifically to address the role of the active-site base (His-452) and its ion pair (Glu-457). Fluorescence spectroscopic data illustrate the role of these amino acids in the function of human dihydrolipoamide dehydrogenase. While mutant H452Q is severely crippled in catalysis of the physiological reaction, the reverse reaction is affected in the E457Q mutant. The K37E mutant shows very little deviation from the wild-type enzyme.
