Mechanism of enhanced melibiose transport rate catalyzed by an Escherichia coli lactose carrier mutant with leucine substituted for serine-306. The pH-dependence of melibiose efflux

Steven King, T. Hastings Wilson

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6 Citations (Scopus)

Abstract

The mechanism of melibiose transport was studied in cells containing plasmid pAA22 which expresses the mutant lactose carrier (serine-306 to leucine) cloned from Escherichia coli AA22. These studies were of interest because several lines of evidence suggested that the AA22 mutation conferred novel properties upon the lactose carrier, decreasing turnover with several β-galactoside substrates, increasing turnover with melibiose, and abolishing active accumulation even though equilibrium occurred via symport with H+. Although severely defective in active melibiose accumulation, the present study indicates that in cells poisoned with azide the AA22 carrier does in fact equilibrate melibiose across the membrane more rapidly than the normal lactose carrier. Similarly, melibiose efflux from cells preloaded with melibiose was more rapidly catalyzed by the AA22 carrier than by the normal carrier (pH 7.0). Furthermore, although external H+ did reduce net melibiose efflux to a rate slower than seen in equilibrium exchange, a lower than normal pH was required to achieve this effect. Therefore, at pH 7.0, the AA22 carrier (but not the normal carrier) catalyzed net efflux at a rate approaching that for the exchange process (which was pH-resistant in both the mutant and the parent). At pH 8.0 both the AA22 carrier and the normal carrier catalyzed net melibiose efflux at a rate indentical to the equilibrium exchange rate. We suggest (i) that the sensitivity of melibiose efflux to external pH indicates that during efflux the AA22 carrier interacts with protons in a manner similar to the normal carrier (i.e., sugar is cotransported with H+) and hence the absence of accumulation is not explained by internal leak via a binary carrier-melibiose complex; and (ii) that the modest increase in rate constants for melibiose exit reflect small changes in activation energy (I kcal/mol) consistent with a steric mechanism possibly involving van der Waals contacts.

Original languageEnglish (US)
Pages (from-to)373-380
Number of pages8
JournalBBA - Biomembranes
Volume1022
Issue number3
DOIs
StatePublished - Mar 16 1990
Externally publishedYes

Fingerprint

Melibiose
Lactose
Leucine
Serine
Escherichia coli
Galactosides
Azides
Ion Transport
Sugars
Protons
Rate constants
Plasmids
Activation energy

Keywords

  • Cotransport
  • Lactose carrier mutant
  • lacY
  • Melibiose efflux
  • pH dependence
  • Proton transport
  • Transport energetics

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology

Cite this

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title = "Mechanism of enhanced melibiose transport rate catalyzed by an Escherichia coli lactose carrier mutant with leucine substituted for serine-306. The pH-dependence of melibiose efflux",
abstract = "The mechanism of melibiose transport was studied in cells containing plasmid pAA22 which expresses the mutant lactose carrier (serine-306 to leucine) cloned from Escherichia coli AA22. These studies were of interest because several lines of evidence suggested that the AA22 mutation conferred novel properties upon the lactose carrier, decreasing turnover with several β-galactoside substrates, increasing turnover with melibiose, and abolishing active accumulation even though equilibrium occurred via symport with H+. Although severely defective in active melibiose accumulation, the present study indicates that in cells poisoned with azide the AA22 carrier does in fact equilibrate melibiose across the membrane more rapidly than the normal lactose carrier. Similarly, melibiose efflux from cells preloaded with melibiose was more rapidly catalyzed by the AA22 carrier than by the normal carrier (pH 7.0). Furthermore, although external H+ did reduce net melibiose efflux to a rate slower than seen in equilibrium exchange, a lower than normal pH was required to achieve this effect. Therefore, at pH 7.0, the AA22 carrier (but not the normal carrier) catalyzed net efflux at a rate approaching that for the exchange process (which was pH-resistant in both the mutant and the parent). At pH 8.0 both the AA22 carrier and the normal carrier catalyzed net melibiose efflux at a rate indentical to the equilibrium exchange rate. We suggest (i) that the sensitivity of melibiose efflux to external pH indicates that during efflux the AA22 carrier interacts with protons in a manner similar to the normal carrier (i.e., sugar is cotransported with H+) and hence the absence of accumulation is not explained by internal leak via a binary carrier-melibiose complex; and (ii) that the modest increase in rate constants for melibiose exit reflect small changes in activation energy (I kcal/mol) consistent with a steric mechanism possibly involving van der Waals contacts.",
keywords = "Cotransport, Lactose carrier mutant, lacY, Melibiose efflux, pH dependence, Proton transport, Transport energetics",
author = "Steven King and Wilson, {T. Hastings}",
year = "1990",
month = "3",
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doi = "10.1016/0005-2736(90)90288-Y",
language = "English (US)",
volume = "1022",
pages = "373--380",
journal = "Biochimica et Biophysica Acta - Biomembranes",
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T1 - Mechanism of enhanced melibiose transport rate catalyzed by an Escherichia coli lactose carrier mutant with leucine substituted for serine-306. The pH-dependence of melibiose efflux

AU - King, Steven

AU - Wilson, T. Hastings

PY - 1990/3/16

Y1 - 1990/3/16

N2 - The mechanism of melibiose transport was studied in cells containing plasmid pAA22 which expresses the mutant lactose carrier (serine-306 to leucine) cloned from Escherichia coli AA22. These studies were of interest because several lines of evidence suggested that the AA22 mutation conferred novel properties upon the lactose carrier, decreasing turnover with several β-galactoside substrates, increasing turnover with melibiose, and abolishing active accumulation even though equilibrium occurred via symport with H+. Although severely defective in active melibiose accumulation, the present study indicates that in cells poisoned with azide the AA22 carrier does in fact equilibrate melibiose across the membrane more rapidly than the normal lactose carrier. Similarly, melibiose efflux from cells preloaded with melibiose was more rapidly catalyzed by the AA22 carrier than by the normal carrier (pH 7.0). Furthermore, although external H+ did reduce net melibiose efflux to a rate slower than seen in equilibrium exchange, a lower than normal pH was required to achieve this effect. Therefore, at pH 7.0, the AA22 carrier (but not the normal carrier) catalyzed net efflux at a rate approaching that for the exchange process (which was pH-resistant in both the mutant and the parent). At pH 8.0 both the AA22 carrier and the normal carrier catalyzed net melibiose efflux at a rate indentical to the equilibrium exchange rate. We suggest (i) that the sensitivity of melibiose efflux to external pH indicates that during efflux the AA22 carrier interacts with protons in a manner similar to the normal carrier (i.e., sugar is cotransported with H+) and hence the absence of accumulation is not explained by internal leak via a binary carrier-melibiose complex; and (ii) that the modest increase in rate constants for melibiose exit reflect small changes in activation energy (I kcal/mol) consistent with a steric mechanism possibly involving van der Waals contacts.

AB - The mechanism of melibiose transport was studied in cells containing plasmid pAA22 which expresses the mutant lactose carrier (serine-306 to leucine) cloned from Escherichia coli AA22. These studies were of interest because several lines of evidence suggested that the AA22 mutation conferred novel properties upon the lactose carrier, decreasing turnover with several β-galactoside substrates, increasing turnover with melibiose, and abolishing active accumulation even though equilibrium occurred via symport with H+. Although severely defective in active melibiose accumulation, the present study indicates that in cells poisoned with azide the AA22 carrier does in fact equilibrate melibiose across the membrane more rapidly than the normal lactose carrier. Similarly, melibiose efflux from cells preloaded with melibiose was more rapidly catalyzed by the AA22 carrier than by the normal carrier (pH 7.0). Furthermore, although external H+ did reduce net melibiose efflux to a rate slower than seen in equilibrium exchange, a lower than normal pH was required to achieve this effect. Therefore, at pH 7.0, the AA22 carrier (but not the normal carrier) catalyzed net efflux at a rate approaching that for the exchange process (which was pH-resistant in both the mutant and the parent). At pH 8.0 both the AA22 carrier and the normal carrier catalyzed net melibiose efflux at a rate indentical to the equilibrium exchange rate. We suggest (i) that the sensitivity of melibiose efflux to external pH indicates that during efflux the AA22 carrier interacts with protons in a manner similar to the normal carrier (i.e., sugar is cotransported with H+) and hence the absence of accumulation is not explained by internal leak via a binary carrier-melibiose complex; and (ii) that the modest increase in rate constants for melibiose exit reflect small changes in activation energy (I kcal/mol) consistent with a steric mechanism possibly involving van der Waals contacts.

KW - Cotransport

KW - Lactose carrier mutant

KW - lacY

KW - Melibiose efflux

KW - pH dependence

KW - Proton transport

KW - Transport energetics

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U2 - 10.1016/0005-2736(90)90288-Y

DO - 10.1016/0005-2736(90)90288-Y

M3 - Article

VL - 1022

SP - 373

EP - 380

JO - Biochimica et Biophysica Acta - Biomembranes

JF - Biochimica et Biophysica Acta - Biomembranes

SN - 0005-2736

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