MONOCLONAL-ANTIBODY 4B1 ALTERS THE PK(A) OF A CARBOXYLIC-ACID AT POSITION-325 (HELIX-X) OF THE LACTOSE PERMEASE OF ESCHERICHIA-COLI

A carboxylic acid al position 325 in helix X is obligatory for lactose /H+ symport at a step corresponding to deprotonation of lactose permea se [Carrasco, N. et al. (1989) Biochemistry 28, 2533-2539]. In this pa per, pH profiles for active transport, efflux, and equilibrium exchang e are analyzed for wild-type permease and mutant Glu325 --> Asp, With respect to active transport and efflux down a concentration gradient, both of which involve net Hf translocation and are defective in the mu tant, the wild-type and the mutant exhibit similar profiles, and at no pH is the mutant stimulated relative to the wild-type, Strikingly, ex change which does not involve H+ translocation is comparable in the wi ld-type and the Glu325 --> Asp mutant below pH 7.5. Above pH 7.5,howev er, the exchange activity of the mutant is progressively and reversibl y inhibited with a midpoint at about pH 8.5; while the exchange activi ty of wild-type permease is only mildly decreased above pH 9.5, and ex change by Glu325 --> Ala or Glu325 --> Gln permease is comparable to w ild-type and unaffected by pH, The findings are consistent with the id ea that translocation of the ternary complex between the permease, lac tose, and H+ does not tolerate a negative charge at position 325. In w ild-type permease, the electrostatic interaction between Glu325 (helix X) and Arg302 (helix IX) is sufficiently strong that the carboxylate is unaffected by pH. Ln contrast, with Asp at position 325, the electr ostatic interaction is broken, the carboxylate becomes protonated, and the acid exhibits a pK(a) of about 8.5, Monoclonal antibody 4B1 binds to the periplasmic loop between helices VII and VIII of the permease [Sun, J. et al. (1996) Biochemistry 35, 990-998] and mimics the Glu325 mutants, Dramatically, 4B1 shifts the apparent pK(a) for exchange fro m about pH 8.5 to 7.5 in the Glu325 --> Asp mutant with little or no e ffect on the wild-type or the Glu325 --> Ala mutant. The findings an c onsistent with the conclusion that the uncoupling effect of 4B1 involv es a conformational change in helix VII and/or VIII chat secondarily a lters the pK(a) of the essential carboxylic acid at position 325.