Steiner P, Fussenegger M, Bailey J E, Sauer U
Institute of Biotechnology, ETH Zürich, CH-8093, Zürich, Switzerland.
Gene. 1998 Oct 5;220(1-2):31-8. doi: 10.1016/s0378-1119(98)00418-1.
The homotetrameric pyruvate kinases (PK) constitute a fine example of allosteric enzymes subjected to sophisticated regulatory mechanisms. We have cloned and sequenced the Zymomonas mobilis structural gene for the first prokaryotic dimeric PK, as an initial step toward understanding the peculiar properties of this enzyme. The deduced amino acid sequence of the pyk gene consists of 475 residues with a calculated molecular mass of 51.4kDa and exhibits up to 50% sequence identity with other PKs. Heterologous expression in Escherichia coli was not obtained from the native promoter, but only when the pyk gene was under the control of a strong inducible promoter when a ribosome-binding site was present upstream of the putative TTG start codon of the pyk gene. Kinetic characterization of PK in concentrated crude cell extracts showed that the enzyme is not activated by sugar phosphates or AMP but is slightly inhibited by ATP. Thus, PK of Z. mobilis is unique among the characterized prokaryotic PKs due to its high activity in the absence of any allosteric activator. Amino acid sequence alignments revealed that glutamate 381 may play a role in ineffective binding of the usual PK activator, fructose-1,6-bisphosphate.
同四聚体丙酮酸激酶（PK）是受到复杂调控机制的变构酶的一个典型例子。我们已经克隆并测序了运动发酵单胞菌中首个原核二聚体PK的结构基因，作为理解该酶特殊性质的第一步。pyk基因推导的氨基酸序列由475个残基组成，计算分子量为51.4 kDa，与其他PK的序列一致性高达50%。在大肠杆菌中，从天然启动子无法实现异源表达，只有当pyk基因处于强诱导型启动子控制下，且在pyk基因假定的TTG起始密码子上游存在核糖体结合位点时才能实现。浓缩粗细胞提取物中PK的动力学特征表明，该酶不受糖磷酸酯或AMP激活，但受到ATP的轻微抑制。因此，运动发酵单胞菌的PK在已表征的原核PK中是独特的，因为它在没有任何变构激活剂的情况下仍具有高活性。氨基酸序列比对显示，谷氨酸381可能在通常的PK激活剂果糖-1,6-二磷酸的无效结合中起作用。
