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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U2 - 10.1074/jbc.274.7.4074
DO - 10.1074/jbc.274.7.4074
M3 - Article
C2 - 9933600
AN - SCOPUS:0033548256
SN - 0021-9258
VL - 274
SP - 4074
EP - 4081
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 7
ER -