THE ROLE OF TYROSINE-114 IN THE ENZYMATIC-ACTIVITY OF THE SHIGA-LIKE TOXIN-I A-CHAIN

被引:15
|
作者
DERESIEWICZ, RL
AUSTIN, PR
HOVDE, CJ
机构
[1] UNIV IDAHO,DEPT BACTERIOL & BIOCHEM,MOSCOW,ID 83843
[2] BRIGHAM & WOMENS HOSP,CHANNING LAB,BOSTON,MA 02115
[3] BRIGHAM & WOMENS HOSP,DEPT MED,DIV INFECT DIS,BOSTON,MA 02115
来源
MOLECULAR & GENERAL GENETICS | 1993年 / 241卷 / 3-4期
关键词
SHIGA-LIKE TOXIN; VEROTOXIN; RICIN; ENTEROTOXIN; ACTIVE SITE;
D O I
10.1007/BF00284701
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
Shiga-like toxin I (SLT-I), the potent cytotoxin produced by certain pathogenic strains of Escherichia coli, is a member of a burgeoning family of ribosome-inactivating proteins (RIPs), which share common structural and mechanistic features. The prototype of the group is the plant toxin ricin. Recently we proposed a structural model for the Slt-IA active site, based in part on the known geometry of the enzymatic subunit of the ricin toxin. The model places three aromatic residues within the putative Slt-IA active site cleft: tyrosine 77, tyrosine 114, and tryptophan 203. Here we present biochemical and biophysical data regarding, the phenotypes of conservative point mutants of Slt-IA in which tyrosine 114 is altered. We used oligonucleotide-directed mutagenesis to replace tyrosine 114 with either phenylalanine (Y114F) or serine (Y114S). Periplasmic extracts of E. coli containing wild-type or mutant Slt-IA were tested for their ability to inhibit protein synthesis in vitro. Relative to wild-type, the activity of mutant Y114F was attenuated about 30-fold, while the mutant Y114S was attenuated about 500 to 1000-fold. In order to address the possibility that differential activation of the mutants rather than local effects at the active site might account for their diminished activity, we engineered the same mutations into a truncated slt-IA cassette that directs expression of a product corresponding to the activated A, form of Slt-IA (wild-type-DELTA). The same general relationships held: relative to wild type-DELTA, Y114F-DELTA was attenuated about 7-fold, and Y114S-DELTA about 300-fold. Tryptic digestion profiles of the mutant proteins were similar to those of the corresponding wild-type, indicating that the amino acid substitutions had not caused major alterations in conformation. We conclude that Y114 plays a significant role in the activity of Slt-IA, one which is quantitatively similar to that of Y77, and one which is predicated on the presence of both its weakly acidic phenolic hydroxyl and its aromatic ring.
引用
收藏
页码:467 / 473
页数:7
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共 33 条
  • [1] MUTATIONS AFFECTING THE ACTIVITY OF THE SHIGA-LIKE TOXIN-I A-CHAIN
    DERESIEWICZ, RL
    CALDERWOOD, SB
    ROBERTUS, JD
    COLLIER, RJ
    [J]. BIOCHEMISTRY, 1992, 31 (12) : 3272 - 3280
  • [2] RAPID DETECTION OF SHIGA TOXIN AND SHIGA-LIKE TOXIN-I
    ASHKENAZI, S
    PICKERING, LK
    CLEARY, TG
    [J]. CLINICAL RESEARCH, 1989, 37 (01): : A58 - A58
  • [3] A METHOD FOR DETECTING SHIGA TOXIN AND SHIGA-LIKE TOXIN-I IN PURE AND MIXED CULTURE
    ASHKENAZI, S
    CLEARY, TG
    [J]. JOURNAL OF MEDICAL MICROBIOLOGY, 1990, 32 (04) : 255 - 261
  • [4] HYBRIDIZATION OF ESCHERICHIA-COLI PRODUCING SHIGA-LIKE TOXIN-I, SHIGA-LIKE TOXIN-II, AND A VARIANT OF SHIGA-LIKE TOXIN-II WITH SYNTHETIC OLIGONUCLEOTIDE PROBES
    BROWN, JE
    SETHABUTR, O
    JACKSON, MP
    LOLEKHA, S
    ECHEVERRIA, P
    [J]. INFECTION AND IMMUNITY, 1989, 57 (09) : 2811 - 2814
  • [5] CRYSTALLIZATION OF THE B-CHAIN OF SHIGA-LIKE TOXIN-I FROM ESCHERICHIA-COLI
    HART, PJ
    MONZINGO, AF
    DONOHUEROLFE, A
    KEUSCH, GT
    CALDERWOOD, SB
    ROBERTUS, JD
    [J]. JOURNAL OF MOLECULAR BIOLOGY, 1991, 218 (04) : 691 - 694
  • [6] EFFECT OF SUBINHIBITORY CONCENTRATIONS OF ANTIBIOTICS ON EXTRACELLULAR SHIGA-LIKE TOXIN-I
    WALTERSPIEL, JN
    ASHKENAZI, S
    MORROW, AL
    CLEARY, TG
    [J]. INFECTION, 1992, 20 (01) : 25 - 29
  • [7] DEVELOPMENT AND EVALUATION OF ENZYME-LINKED IMMUNOSORBENT ASSAYS FOR DETECTION OF SHIGA-LIKE TOXIN-I AND SHIGA-LIKE TOXIN-II
    DOWNES, FP
    GREEN, JH
    GREENE, K
    STROCKBINE, N
    WELLS, JG
    WACHSMUTH, IK
    [J]. JOURNAL OF CLINICAL MICROBIOLOGY, 1989, 27 (06) : 1292 - 1297
  • [8] DEVELOPMENT OF SANDWICH HYBRIDIZATION ASSAY FOR THE DETECTION OF SHIGA-LIKE TOXIN-I AND SHIGA-LIKE TOXIN-II DETERMINANTS IN ESCHERICHIA-COLI
    BIALKOWSKAHOBRZANSKA, H
    FLETCHER, RF
    HURLEY, M
    GREGSON, DB
    HAMMERBERG, O
    [J]. MEDICAL MICROBIOLOGY LETTERS, 1993, 2 (08): : 411 - 418
  • [9] DNA PROBES FOR SHIGA-LIKE TOXIN-I AND TOXIN-II AND FOR TOXIN-CONVERTING BACTERIOPHAGES
    NEWLAND, JW
    NEILL, RJ
    [J]. JOURNAL OF CLINICAL MICROBIOLOGY, 1988, 26 (07) : 1292 - 1297
  • [10] EFFECTS OF HUMAN INTRAVENOUS IMMUNE GLOBULIN ON DIARRHEA CAUSED BY SHIGA-LIKE TOXIN-I AND SHIGA-LIKE TOXIN-II IN INFANT RABBITS
    HAVENS, PL
    DUNNE, WM
    BURD, EM
    [J]. MICROBIOLOGY AND IMMUNOLOGY, 1992, 36 (10) : 1077 - 1085