Monoclonal Antibody 4B1 Alters the pKa of a Carboxylic Acid at Position 325 (Helix X) of the Lactose Permease of Escherichia coli
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- 作者：Stathis Frillingos and H. Ronald Kaback
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：August 6, 1996
- 年：1996
- 卷：35
- 期：31
- 页码：10166 - 10171
- 全文大小：342K
- 年卷期：v.35,no.31(August 6, 1996)
- ISSN：1520-4995
文摘
A carboxylic acid at position 325 in helix X isobligatory for lactose/H+ symport at a stepcorresponding to deprotonation of lactose permease [Carrasco, N. etal. (1989) Biochemistry 28, 2533-2539]. In this paper, pH profiles for active transport, efflux,and equilibrium exchange are analyzed forwild-type permease and mutant Glu325 
Asp. With respect toactive transport and efflux down aconcentration gradient, both of which involve net H+translocation and are defective in the mutant, thewild-type and the mutant exhibit similar profiles, and at no pH is themutant stimulated relative to thewild-type. Strikingly, exchange which does not involveH+ translocation is comparable in thewild-typeand the Glu325 Asp mutant below pH 7.5. Above pH 7.5, however,the exchange activity of themutant is progressively and reversibly inhibited with a midpoint atabout pH 8.5; while the exchangeactivity of wild-type permease is only mildly decreased above pH 9.5,and exchange by Glu325 Alaor Glu325 Gln permease is comparable to wild-type and unaffected bypH. The findings are consistentwith the idea that translocation of the ternary complex between thepermease, lactose, and H+ does nottolerate a negative charge at position 325. In wild-type permease,the electrostatic interaction betweenGlu325 (helix X) and Arg302 (helix IX) is sufficiently strong that thecarboxylate is unaffected by pH.In contrast, with Asp at position 325, the electrostaticinteraction is broken, the carboxylate becomesprotonated, and the acid exhibits a pKa of about8.5. Monoclonal antibody 4B1 binds to the periplasmicloop between helices VII and VIII of the permease [Sun, J. et al.(1996) Biochemistry 35, 990-998]andmimics the Glu325 mutants. Dramatically, 4B1 shifts the apparentpKa for exchange from about pH 8.5to 7.5 in the Glu325 Asp mutant with little or no effect on thewild-type or the Glu325 Ala mutant.The findings are consistent with the conclusion that theuncoupling effect of 4B1 involves a conformationalchange in helix VII and/or VIII that secondarily alters thepKa of the essential carboxylic acid atposition325.
Asp. With respect toactive transport and efflux down aconcentration gradient, both of which involve net H+translocation and are defective in the mutant, thewild-type and the mutant exhibit similar profiles, and at no pH is themutant stimulated relative to thewild-type. Strikingly, exchange which does not involveH+ translocation is comparable in thewild-typeand the Glu325 Asp mutant below pH 7.5. Above pH 7.5, however,the exchange activity of themutant is progressively and reversibly inhibited with a midpoint atabout pH 8.5; while the exchangeactivity of wild-type permease is only mildly decreased above pH 9.5,and exchange by Glu325 Alaor Glu325 Gln permease is comparable to wild-type and unaffected bypH. The findings are consistentwith the idea that translocation of the ternary complex between thepermease, lactose, and H+ does nottolerate a negative charge at position 325. In wild-type permease,the electrostatic interaction betweenGlu325 (helix X) and Arg302 (helix IX) is sufficiently strong that thecarboxylate is unaffected by pH.In contrast, with Asp at position 325, the electrostaticinteraction is broken, the carboxylate becomesprotonated, and the acid exhibits a pKa of about8.5. Monoclonal antibody 4B1 binds to the periplasmicloop between helices VII and VIII of the permease [Sun, J. et al.(1996) Biochemistry 35, 990-998]andmimics the Glu325 mutants. Dramatically, 4B1 shifts the apparentpKa for exchange from about pH 8.5to 7.5 in the Glu325 Asp mutant with little or no effect on thewild-type or the Glu325 Ala mutant.The findings are consistent with the conclusion that theuncoupling effect of 4B1 involves a conformationalchange in helix VII and/or VIII that secondarily alters thepKa of the essential carboxylic acid atposition325.