Jung K, Altendorf K
Universität Osnabrück, Fachbereich Biologie/Chemie, Abteilung Mikrobiologie, D-49069 Osnabrück, Germany.
J Biol Chem. 1998 Oct 9;273(41):26415-20. doi: 10.1074/jbc.273.41.26415.
Escherichia coli responds to K+ limitation or high osmolarity by induction of the kdpFABC operon coding for the high affinity K+-translocating Kdp-ATPase. KdpD, the sensor kinase of this system, is a bifunctional enzyme catalyzing the autophosphorylation by ATP and the dephosphorylation of the corresponding response regulator KdpE. Here we demonstrate that individual replacements of clustered arginine residues located close to transmembrane domain TM4 modulate the ratio of kinase to phosphatase activity. Thus KdpD-Arg511 --> Gln is characterized by an increase in the kinase activity and a loss of the phosphatase activity. However, when Arg at position 511 is replaced with Lys, activities of the corresponding protein are comparable with wild-type KdpD. In contrast, replacement of arginine residues at positions 503, 506, or 508 with glutamine or lysine causes a decrease of the kinase and an increase of the phosphatase activities. Changes of the activities of these KdpD proteins correspond with alterations in kdpFABC expression. Thus KdpD-Arg511 --> Gln causes constitutive expression of kdpFABC. KdpD proteins with Arg replacements at positions 503, 506, or 508 are unable to respond to osmolarity, whereas the sensing of K+ limitation is not influenced. Simultaneous replacement of arginine residues 508 and 511 or 506, 508, and 511 with glutamine leads to a decrease of the phosphatase activity. However, kdpFABC expression is dependent on K+ and osmolarity. Finally, when Arg513 is replaced with glutamine the amount of KdpD detected in the membrane is drastically reduced. These results imply that there is an equilibrium between the kinase and phosphatase activities of KdpD, which can be shifted by the replacement of one arginine residue. An electrostatic switch mechanism within the protein is proposed through which the ratio of kinase to phosphatase is regulated. Finally, these results lend support to the notion that KdpD can be activated by two distinct stimuli, K+ limitation and osmolarity.
大肠杆菌通过诱导编码高亲和力钾转运Kdp - ATP酶的kdpFABC操纵子来响应钾离子限制或高渗透压。该系统的传感激酶KdpD是一种双功能酶，催化ATP的自磷酸化以及相应反应调节因子KdpE的去磷酸化。在此我们证明，靠近跨膜结构域TM4的成簇精氨酸残基的个别替换会调节激酶与磷酸酶活性的比例。因此，KdpD - Arg511→Gln的特征是激酶活性增加而磷酸酶活性丧失。然而，当511位的精氨酸被赖氨酸取代时，相应蛋白的活性与野生型KdpD相当。相反，用谷氨酰胺或赖氨酸取代503、506或508位的精氨酸残基会导致激酶活性降低和磷酸酶活性增加。这些KdpD蛋白活性的变化与kdpFABC表达的改变相对应。因此，KdpD - Arg511→Gln导致kdpFABC的组成型表达。在503、506或508位被精氨酸取代的KdpD蛋白无法响应渗透压，而对钾离子限制的感知不受影响。将508和511位或506、508和511位的精氨酸残基同时替换为谷氨酰胺会导致磷酸酶活性降低。然而，kdpFABC的表达依赖于钾离子和渗透压。最后，当513位的精氨酸被谷氨酰胺取代时，在膜中检测到的KdpD量大幅减少。这些结果表明，KdpD的激酶和磷酸酶活性之间存在平衡，这种平衡可通过一个精氨酸残基的替换而改变。我们提出了一种蛋白质内的静电开关机制，通过该机制调节激酶与磷酸酶的比例。最后，这些结果支持了KdpD可被两种不同刺激（钾离子限制和渗透压）激活的观点。
