THE MALTOSE TRANSPORT-SYSTEM OF ESCHERICHIA-COLI DISPLAYS POSITIVE COOPERATIVITY IN ATP HYDROLYSIS

Maltose transport across the cytoplasmic membrane of Escherichia coil is catalyzed by a periplasmic binding protein-dependent transport syst em and energized by ATP. The maltose system, a member of the ATP-bindi ng cassette or ABC transport family, contains two copies of an ATP-bin ding protein in a complex with two integral membrane proteins. ATP hyd rolysis by the transport complex can be assayed following reconstituti on into proteoliposomes in the presence of maltose binding protein and maltose. Mutations in the transport complex that permit binding prote in-independent transport render ATP hydrolysis constitutive so that hy drolysis can also be assayed with the transport complex in detergent s olution. We have used both of these systems to study the role of two A TP binding sites in ATP hydrolysis. We found that both the wild-type a nd the binding protein-independent systems hydrolyzed ATP with positiv e cooperativity, suggesting that the two ATP binding sites interact. V anadate inhibited the ATPase activity of the transport complex with 50 % inhibition occurring at 10 mu M vanadate. In detergent solution, the degree of cooperativity in the binding protein-independent complex de creased with increasing pH. The loss of cooperativity was accompanied by a decrease in ATPase activity and a decrease in sensitivity to vana date. Because reconstitution of the complex into a lipid bilayer preve nted the loss of cooperativity, we expect that ATP hydrolysis is coope rative in vivo. The mutations leading to binding protein-independent t ransport do not significantly alter the affinity, cooperativity, vanad ate sensitivity, or substrate specificity of the ATP binding sites dur ing hydrolysis. These results justify the use of the binding protein-i ndependent system to investigate the mechanism of transport and hydrol ysis.