Vieille Claire, Krishnamurthy Harini, Hyun Hyung-Hwan, Savchenko Alexei, Yan Honggao, Zeikus J Gregory
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing 48824, USA.
Biochem J. 2003 Jun 1;372(Pt 2):577-85. doi: 10.1042/BJ20021377.
The adenylate kinase (AK) gene from Thermotoga neapolitana, a hyperthermophilic bacterium, was cloned and overexpressed in Escherichia coli, and the recombinant enzyme was biochemically characterized. The T. neapolitana AK (TNAK) sequence indicates that this enzyme belongs to the long bacterial AKs. TNAK contains the four cysteine residues that bind Zn(2+) in all Gram-positive AKs and in a few other Zn(2+)-containing bacterial AKs. Atomic emission spectroscopy and titration data indicate a content of 1 mol of Zn(2+)/mol of recombinant TNAK. The EDTA-treated enzyme has a melting temperature (T (m)=93.5 degrees C) 6.2 degrees C below that of the holoenzyme (99.7 degrees C), identifying Zn(2+) as a stabilizing feature in TNAK. TNAK is a monomeric enzyme with a molecular mass of approx. 25 kDa. TNAK displays V (max) and K (m) values at 30 degrees C identical with those of the E. coli AK at 30 degrees C, and displays very high activity at 80 degrees C, with a specific activity above 8000 units/mg. The unusually high activity of TNAK at 30 degrees C makes it an interesting model to test the role of enzyme flexibility in activity.
克隆了嗜热栖热菌（一种嗜热细菌）的腺苷酸激酶（AK）基因，并在大肠杆菌中进行了过量表达，对重组酶进行了生化特性分析。嗜热栖热菌AK（TNAK）序列表明该酶属于长链细菌AKs。TNAK含有在所有革兰氏阳性AKs以及其他一些含Zn(2+)的细菌AKs中结合Zn(2+)的四个半胱氨酸残基。原子发射光谱和滴定数据表明重组TNAK中Zn(2+)的含量为1摩尔/摩尔。经EDTA处理的酶的解链温度（T (m)=93.5℃）比全酶（99.7℃）低6.2℃，这表明Zn(2+)是TNAK的一个稳定特征。TNAK是一种分子量约为25 kDa的单体酶。TNAK在30℃时的V (max)和K (m)值与大肠杆菌AK在30℃时相同，并且在80℃时具有非常高的活性，比活性超过8000单位/毫克。TNAK在30℃时异常高的活性使其成为测试酶灵活性在活性中作用的一个有趣模型。
