Details
| Original language | English |
|---|---|
| Pages (from-to) | 5682-8 |
| Number of pages | 7 |
| Journal | Biochemistry |
| Volume | 37 |
| Issue number | 16 |
| Publication status | Published - 21 Apr 1998 |
| Externally published | Yes |
Abstract
EcoRV is a dimer of two identical subunits which together form one binding site for the double-stranded DNA substrate. Concerted cleavage of both strands of the duplex requires intersubunit communication to synchronize the two catalytic centers of EcoRV. Here we address the question of how contacts to the DNA backbone trigger conformational changes which lead to the activation of both catalytic centers. The structure of the specific EcoRV-DNA complex shows that a region including amino acids Thr 37 and Lys 38 is involved in interactions with the DNA backbone and is a candidate for intersubunit communication. Homodimeric EcoRV T37A and K38A variants have a 1000-fold reduced catalytic activity. To examine whether Thr 37 and Lys 38 of one subunit affect the catalytic center in the same subunit and/or in the other subunit, we have produced heterodimeric variants containing a Thr 37 --> Ala or Lys 38 --> Ala substitution in one subunit combined with a wild type (wt) subunit (wt/T37A and wt/K38A) or with a subunit which contains an amino acid substitution (Asp 90 --> Ala) in the active site (D90A/T37A and D90A/K38A). Cleavage experiments with supercoiled pAT153 show that wt/T37A and wt/K38A preferentially nick the DNA. A steady-state kinetic analysis of the cleavage of an oligodeoxynucleotide substrate shows that the activity of wt/T37A and wt/K38A is half of that of wild type EcoRV, whereas D90A/T37A and D90A/K38A are almost inactive. These results demonstrate that Thr 37 and Lys 38 affect primarily the catalytic center in their own subunit and that both subunits of EcoRV can be activated independently of each other. We suggest that Thr 37 and Lys 38 control the catalytic activity of the active site in their own subunit by positioning alpha-helix B.
Keywords
- Catalysis, Crystallography, X-Ray, DNA, Bacterial/metabolism, Deoxyribonucleases, Type II Site-Specific/genetics, Dimerization, Electrophoresis, Polyacrylamide Gel, Escherichia coli/enzymology, Lysine/genetics, Models, Molecular, Mutagenesis, Site-Directed, Oligodeoxyribonucleotides/metabolism, Plasmids/metabolism, Threonine/metabolism
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In: Biochemistry, Vol. 37, No. 16, 21.04.1998, p. 5682-8.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Intra- vs intersubunit communication in the homodimeric restriction enzyme EcoRV
T2 - Thr 37 and Lys 38 involved in indirect readout are only important for the catalytic activity of their own subunit
AU - Stahl, F
AU - Wende, W
AU - Wenz, C
AU - Jeltsch, A
AU - Pingoud, A
PY - 1998/4/21
Y1 - 1998/4/21
N2 - EcoRV is a dimer of two identical subunits which together form one binding site for the double-stranded DNA substrate. Concerted cleavage of both strands of the duplex requires intersubunit communication to synchronize the two catalytic centers of EcoRV. Here we address the question of how contacts to the DNA backbone trigger conformational changes which lead to the activation of both catalytic centers. The structure of the specific EcoRV-DNA complex shows that a region including amino acids Thr 37 and Lys 38 is involved in interactions with the DNA backbone and is a candidate for intersubunit communication. Homodimeric EcoRV T37A and K38A variants have a 1000-fold reduced catalytic activity. To examine whether Thr 37 and Lys 38 of one subunit affect the catalytic center in the same subunit and/or in the other subunit, we have produced heterodimeric variants containing a Thr 37 --> Ala or Lys 38 --> Ala substitution in one subunit combined with a wild type (wt) subunit (wt/T37A and wt/K38A) or with a subunit which contains an amino acid substitution (Asp 90 --> Ala) in the active site (D90A/T37A and D90A/K38A). Cleavage experiments with supercoiled pAT153 show that wt/T37A and wt/K38A preferentially nick the DNA. A steady-state kinetic analysis of the cleavage of an oligodeoxynucleotide substrate shows that the activity of wt/T37A and wt/K38A is half of that of wild type EcoRV, whereas D90A/T37A and D90A/K38A are almost inactive. These results demonstrate that Thr 37 and Lys 38 affect primarily the catalytic center in their own subunit and that both subunits of EcoRV can be activated independently of each other. We suggest that Thr 37 and Lys 38 control the catalytic activity of the active site in their own subunit by positioning alpha-helix B.
AB - EcoRV is a dimer of two identical subunits which together form one binding site for the double-stranded DNA substrate. Concerted cleavage of both strands of the duplex requires intersubunit communication to synchronize the two catalytic centers of EcoRV. Here we address the question of how contacts to the DNA backbone trigger conformational changes which lead to the activation of both catalytic centers. The structure of the specific EcoRV-DNA complex shows that a region including amino acids Thr 37 and Lys 38 is involved in interactions with the DNA backbone and is a candidate for intersubunit communication. Homodimeric EcoRV T37A and K38A variants have a 1000-fold reduced catalytic activity. To examine whether Thr 37 and Lys 38 of one subunit affect the catalytic center in the same subunit and/or in the other subunit, we have produced heterodimeric variants containing a Thr 37 --> Ala or Lys 38 --> Ala substitution in one subunit combined with a wild type (wt) subunit (wt/T37A and wt/K38A) or with a subunit which contains an amino acid substitution (Asp 90 --> Ala) in the active site (D90A/T37A and D90A/K38A). Cleavage experiments with supercoiled pAT153 show that wt/T37A and wt/K38A preferentially nick the DNA. A steady-state kinetic analysis of the cleavage of an oligodeoxynucleotide substrate shows that the activity of wt/T37A and wt/K38A is half of that of wild type EcoRV, whereas D90A/T37A and D90A/K38A are almost inactive. These results demonstrate that Thr 37 and Lys 38 affect primarily the catalytic center in their own subunit and that both subunits of EcoRV can be activated independently of each other. We suggest that Thr 37 and Lys 38 control the catalytic activity of the active site in their own subunit by positioning alpha-helix B.
KW - Catalysis
KW - Crystallography, X-Ray
KW - DNA, Bacterial/metabolism
KW - Deoxyribonucleases, Type II Site-Specific/genetics
KW - Dimerization
KW - Electrophoresis, Polyacrylamide Gel
KW - Escherichia coli/enzymology
KW - Lysine/genetics
KW - Models, Molecular
KW - Mutagenesis, Site-Directed
KW - Oligodeoxyribonucleotides/metabolism
KW - Plasmids/metabolism
KW - Threonine/metabolism
U2 - 10.1021/bi973025s
DO - 10.1021/bi973025s
M3 - Article
C2 - 9548954
VL - 37
SP - 5682
EP - 5688
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 16
ER -