A K319N/E325Q double mutant of the lactose permease cotransports H+ with lactose: Implications for a proposed mechanism of H+/lactose symport

Jerry L. Johnson, Robert J Brooker

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18 Citations (Scopus)

Abstract

In this study, we have examined the transport characteristics of the wild-type lactose permease, single mutants in which Lys-319 was changed to asparagine or alanine or Glu-325 was changed to glutamine or alanine, and the corresponding double mutant strains. The wild-type and Asn-319 mutant showed high levels of lactose uptake, with K(m) values of 0.42 and 1.30 mM and V(max) values of 102.6 and 48.3 nmol of lactose/min/mg of protein, respectively. The Asn-319/Gln-325 strain had a normal K(m) of 0.36 mM and a moderate V(max) of 18.5 nmol of lactose/min/mg of protein. By comparison, the single E325Q strain had a normal K(m) of 0.27 mM but a very defective V(max) of 1.3 nmol of lactose/min/mg of protein. A similar trend was observed among the alanine substitutions at these positions, although the V(max) values were lower for the Ala-319 mutations. When comparing the V(max) values between the single position 325 mutants with those of the double mutants, these results indicate that neutral 319 mutations substantially alleviate a defect in V(max) caused by neutral 325 mutations. With regard to H+/lactose coupling, the wild-type permease is normally coupled and can transport lactose against a gradient. The position 325 single mutants showed no evidence of H+ transport with lactose or thiodigalactoside (TDG) and were unable to facilitate uphill lactose transport. The single Asn-319 mutant and double Asn-319/Gln-325 mutant were able to transport H+ upon the addition of lactose or TDG. In addition, both of these strains catalyzed a sugar-dependent H+ leak that inhibited cell growth in the presence of TDG. These two strains were also defective in uphill transport, which may be related to their sugar- dependent leak pathway. Based on these and other results in the literature, a model is presented that describes how the interactions among several ionizable residues within the lactose permease act in a concerted manner to control H+/lactose coupling. In this model, Lys-319 and Glu-325 play a central role in governing the ability of the lactose permease to couple the transport of H+ and lactose.

Original languageEnglish (US)
Pages (from-to)4074-4081
Number of pages8
JournalJournal of Biological Chemistry
Volume274
Issue number7
DOIs
StatePublished - Feb 12 1999

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Ion Transport
Lactose
Alanine
Active Biological Transport
Sugars
Mutation
lactose permease
Proteins
Membrane Transport Proteins
Asparagine
Cell growth
Glutamine
Substitution reactions
Defects

Cite this

@article{7dc875112d2b478e9cdcea7a5301d08f,
title = "A K319N/E325Q double mutant of the lactose permease cotransports H+ with lactose: Implications for a proposed mechanism of H+/lactose symport",
abstract = "In this study, we have examined the transport characteristics of the wild-type lactose permease, single mutants in which Lys-319 was changed to asparagine or alanine or Glu-325 was changed to glutamine or alanine, and the corresponding double mutant strains. The wild-type and Asn-319 mutant showed high levels of lactose uptake, with K(m) values of 0.42 and 1.30 mM and V(max) values of 102.6 and 48.3 nmol of lactose/min/mg of protein, respectively. The Asn-319/Gln-325 strain had a normal K(m) of 0.36 mM and a moderate V(max) of 18.5 nmol of lactose/min/mg of protein. By comparison, the single E325Q strain had a normal K(m) of 0.27 mM but a very defective V(max) of 1.3 nmol of lactose/min/mg of protein. A similar trend was observed among the alanine substitutions at these positions, although the V(max) values were lower for the Ala-319 mutations. When comparing the V(max) values between the single position 325 mutants with those of the double mutants, these results indicate that neutral 319 mutations substantially alleviate a defect in V(max) caused by neutral 325 mutations. With regard to H+/lactose coupling, the wild-type permease is normally coupled and can transport lactose against a gradient. The position 325 single mutants showed no evidence of H+ transport with lactose or thiodigalactoside (TDG) and were unable to facilitate uphill lactose transport. The single Asn-319 mutant and double Asn-319/Gln-325 mutant were able to transport H+ upon the addition of lactose or TDG. In addition, both of these strains catalyzed a sugar-dependent H+ leak that inhibited cell growth in the presence of TDG. These two strains were also defective in uphill transport, which may be related to their sugar- dependent leak pathway. Based on these and other results in the literature, a model is presented that describes how the interactions among several ionizable residues within the lactose permease act in a concerted manner to control H+/lactose coupling. In this model, Lys-319 and Glu-325 play a central role in governing the ability of the lactose permease to couple the transport of H+ and lactose.",
author = "Johnson, {Jerry L.} and Brooker, {Robert J}",
year = "1999",
month = "2",
day = "12",
doi = "10.1074/jbc.274.7.4074",
language = "English (US)",
volume = "274",
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journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
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TY - JOUR

T1 - A K319N/E325Q double mutant of the lactose permease cotransports H+ with lactose

T2 - Implications for a proposed mechanism of H+/lactose symport

AU - Johnson, Jerry L.

AU - Brooker, Robert J

PY - 1999/2/12

Y1 - 1999/2/12

N2 - In this study, we have examined the transport characteristics of the wild-type lactose permease, single mutants in which Lys-319 was changed to asparagine or alanine or Glu-325 was changed to glutamine or alanine, and the corresponding double mutant strains. The wild-type and Asn-319 mutant showed high levels of lactose uptake, with K(m) values of 0.42 and 1.30 mM and V(max) values of 102.6 and 48.3 nmol of lactose/min/mg of protein, respectively. The Asn-319/Gln-325 strain had a normal K(m) of 0.36 mM and a moderate V(max) of 18.5 nmol of lactose/min/mg of protein. By comparison, the single E325Q strain had a normal K(m) of 0.27 mM but a very defective V(max) of 1.3 nmol of lactose/min/mg of protein. A similar trend was observed among the alanine substitutions at these positions, although the V(max) values were lower for the Ala-319 mutations. When comparing the V(max) values between the single position 325 mutants with those of the double mutants, these results indicate that neutral 319 mutations substantially alleviate a defect in V(max) caused by neutral 325 mutations. With regard to H+/lactose coupling, the wild-type permease is normally coupled and can transport lactose against a gradient. The position 325 single mutants showed no evidence of H+ transport with lactose or thiodigalactoside (TDG) and were unable to facilitate uphill lactose transport. The single Asn-319 mutant and double Asn-319/Gln-325 mutant were able to transport H+ upon the addition of lactose or TDG. In addition, both of these strains catalyzed a sugar-dependent H+ leak that inhibited cell growth in the presence of TDG. These two strains were also defective in uphill transport, which may be related to their sugar- dependent leak pathway. Based on these and other results in the literature, a model is presented that describes how the interactions among several ionizable residues within the lactose permease act in a concerted manner to control H+/lactose coupling. In this model, Lys-319 and Glu-325 play a central role in governing the ability of the lactose permease to couple the transport of H+ and lactose.

AB - In this study, we have examined the transport characteristics of the wild-type lactose permease, single mutants in which Lys-319 was changed to asparagine or alanine or Glu-325 was changed to glutamine or alanine, and the corresponding double mutant strains. The wild-type and Asn-319 mutant showed high levels of lactose uptake, with K(m) values of 0.42 and 1.30 mM and V(max) values of 102.6 and 48.3 nmol of lactose/min/mg of protein, respectively. The Asn-319/Gln-325 strain had a normal K(m) of 0.36 mM and a moderate V(max) of 18.5 nmol of lactose/min/mg of protein. By comparison, the single E325Q strain had a normal K(m) of 0.27 mM but a very defective V(max) of 1.3 nmol of lactose/min/mg of protein. A similar trend was observed among the alanine substitutions at these positions, although the V(max) values were lower for the Ala-319 mutations. When comparing the V(max) values between the single position 325 mutants with those of the double mutants, these results indicate that neutral 319 mutations substantially alleviate a defect in V(max) caused by neutral 325 mutations. With regard to H+/lactose coupling, the wild-type permease is normally coupled and can transport lactose against a gradient. The position 325 single mutants showed no evidence of H+ transport with lactose or thiodigalactoside (TDG) and were unable to facilitate uphill lactose transport. The single Asn-319 mutant and double Asn-319/Gln-325 mutant were able to transport H+ upon the addition of lactose or TDG. In addition, both of these strains catalyzed a sugar-dependent H+ leak that inhibited cell growth in the presence of TDG. These two strains were also defective in uphill transport, which may be related to their sugar- dependent leak pathway. Based on these and other results in the literature, a model is presented that describes how the interactions among several ionizable residues within the lactose permease act in a concerted manner to control H+/lactose coupling. In this model, Lys-319 and Glu-325 play a central role in governing the ability of the lactose permease to couple the transport of H+ and lactose.

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