CYSTEINE-148 IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI IS A COMPONENT OF A SUBSTRATE-BINDING SITE .1. SITE-DIRECTED MUTAGENESIS STUDIES

被引:59
作者
JUNG, H
JUNG, K
KABACK, HR
机构
[1] UNIV CALIF LOS ANGELES, INST MOLEC BIOL, HOWARD HUGHES MED INST, DEPT PHYSIOL, LOS ANGELES, CA 90024 USA
[2] UNIV CALIF LOS ANGELES, INST MOLEC BIOL, HOWARD HUGHES MED INST, DEPT MICROBIOL & MOLEC GENET, LOS ANGELES, CA 90024 USA
关键词
D O I
10.1021/bi00206a019
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Cys148 in the lactose permease of Escherichia coli has been replaced with hydrophobic (Ala, Val, Ile, Phe), hydrophilic (Ser, Thr), or charged (Asp, Lys) residues, and the properties of the replacement mutants have been analyzed. Although Cys148 is not essential for transport, the size and polarity of the side chain at this position modifies transport activity and substrate specificity. Thus, small hydrophobic side chains (Ala, Val) generally increase the apparent affinity of the permease for substrate, while hydrophilic side chains (Ser, Thr, Asp) decrease apparent affinity and bulky or positively charged side chains (Phe, Lys) virtually abolish activity. In addition, hydrophilic substitutions (Ser, Thr, Asp) alter the specificity of the permease toward monosaccharides relative to disaccharides. On the basis of these and other observations, it is concluded that Cys148 is located in a sugar binding site of lac permease and probably interacts hydrophobically with the galactosyl moiety. The postulate receives more direct support from site-directed fluorescence labeling studies presented in the following paper in this issue [Wu, J., and Kaback, H. R. (1994) Biochemistry (following paper in this issue)].
引用
收藏
页码:12160 / 12165
页数:6
相关论文
共 45 条
[1]  
BEYREUTHER K, 1981, METHODS PROTEIN SEQU, P139
[2]   A COMPLEMENTATION ANALYSIS OF RESTRICTION AND MODIFICATION OF DNA IN ESCHERICHIA COLI [J].
BOYER, HW ;
ROULLAND.D .
JOURNAL OF MOLECULAR BIOLOGY, 1969, 41 (03) :459-&
[3]  
BROOKER RJ, 1986, J BIOL CHEM, V261, P1765
[4]   LAC PERMEASE OF ESCHERICHIA-COLI - TOPOLOGY AND SEQUENCE ELEMENTS PROMOTING MEMBRANE INSERTION [J].
CALAMIA, J ;
MANOIL, C .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1990, 87 (13) :4937-4941
[5]   INTRAMOLECULAR DISLOCATION OF THE COOH TERMINUS OF THE LAC CARRIER PROTEIN IN RECONSTITUTED PROTEOLIPOSOMES [J].
CARRASCO, N ;
HERZLINGER, D ;
MITCHELL, R ;
DECHIARA, S ;
DANHO, W ;
GABRIEL, TF ;
KABACK, HR .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES, 1984, 81 (15) :4672-4676
[6]   ROLE OF PROLINE RESIDUES IN THE STRUCTURE AND FUNCTION OF A MEMBRANE-TRANSPORT PROTEIN [J].
CONSLER, TG ;
TSOLAS, O ;
KABACK, HR .
BIOCHEMISTRY, 1991, 30 (05) :1291-1298
[7]  
FOSTER DL, 1983, J BIOL CHEM, V258, P31
[9]   DIDEOXY SEQUENCING METHOD USING DENATURED PLASMID TEMPLATES [J].
HATTORI, M ;
SAKAKI, Y .
ANALYTICAL BIOCHEMISTRY, 1986, 152 (02) :232-238
[10]   FUNCTIONAL AND IMMUNOCHEMICAL CHARACTERIZATION OF A MUTANT OF ESCHERICHIA-COLI ENERGY UNCOUPLED FOR LACTOSE TRANSPORT [J].
HERZLINGER, D ;
CARRASCO, N ;
KABACK, HR .
BIOCHEMISTRY, 1985, 24 (01) :221-229