INDIVIDUAL SUBSTITUTIONS OF CLUSTERED ARGININE RESIDUES OF THE SENSORKINASE KDPD OF ESCHERICHIA-COLI MODULATE THE RATIO OF KINASE TO PHOSPHATASE-ACTIVITY

Escherichia coli responds to K+ limitation or high osmolarity by induc tion of the kdpFABC operon coding for the high affinity K+-translocati ng Kdp-ATPase, KdpD, the sensor kinase of this system, is a bifunction al enzyme catalyzing the autophosphorylation by ATP and the dephosphor ylation of the corresponding response regulator KdpE, Here we demonstr ate that individual replacements of clustered arginine residues locate d close to transmembrane domain TM4 modulate the ratio of kinase to ph osphatase activity. Thus KdpD-Arg(511) --> 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 o f the corresponding protein are comparable with wildtype KdpD, In cont rast, replacement of arginine residues at positions 503, 506, or 508 w ith glutamine or lysine causes a decrease of the kinase and an increas e of the phosphatase activities. Changes of the activities of these Kd pD proteins correspond with alterations in kdpFABC expression. Thus Kd pD-Arg511 --> Gin causes constitutive expression of kdpFABC. KdpD prot eins with Arg replacements at positions 503, 506, or 508 are unable to respond to osmolarity, whereas the sensing of K+ limitation is not in fluenced. Simultaneous replacement of arginine residues 508 and 511 or 506, 508, and 511 with glutamine leads to a decrease of the phosphata se activity. However, kdpFABC expression is dependent on K+ and osmola rity, Finally, when Arg(513) is replaced with glutamine the amount of KdpD detected in the membrane is drastically reduced. These results im ply that there is an equilibrium between the kinase and phosphatase ac tivities of KdpD, which can be shifted by the replacement of one argin ine residue. An electrostatic switch mechanism within the protein is p roposed through which the ratio of kinase to phosphatase is regulated. Finally, these results lend support to the notion that KdpD can be ac tivated by two distinct stimuli, K+ limitation and osmolarity.