MUTATIONAL ANALYSIS OF THE FUNCTION OF GLN115 IN THE ECORI RESTRICTION ENDONUCLEASE, A CRITICAL AMINO-ACID FOR RECOGNITION OF THE INNER THYMIDINE RESIDUE IN THE SEQUENCE-GAATTC AND FOR COUPLING SPECIFIC DNA-BINDING TO CATALYSIS

被引:32
作者
JELTSCH, A
ALVES, J
OELGESCHLAGER, T
WOLFES, H
MAASS, G
PINGOUD, A
机构
[1] Inst. F. Biophysikalische Chemie, Zentrum Biochemie, Medizinische Hochschule Hannover, D-3000 Hannover 61
关键词
DNA; RESTRICTION ENZYME; HYDROPHOBIC INTERACTION; SITE-DIRECTED MUTAGENESIS; SPECIFICITY;
D O I
10.1006/jmbi.1993.1019
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
The Gln115 residue of the EcoRI restriction endonuclease has been proposed to form a hydrophobic contact to the methyl group of the inner thymidine of the EcoRI recognition sequence -GAATTC- and to be involved in intramolecular hydrogen bonds to the main-chain at positions 140 and 143 as well as to the side-chain of Asn173. We have exchanged Gln115 for Ala and Glu by site-directed mutagenesis and analysed the purified mutant proteins (Q115A and Q115E) biochemically and physico-chemically. Q115A and Q115E have the same secondary structure composition as wild-type EcoRI but are less stable towards thermal denaturation than the wild-type enzyme. In contrast to wild-type EcoRI the mutant proteins show a biphasic denaturation profile under alkaline pH, presumably because the amino acid exchange labilizes one part of the molecule, which unfolds before the rest of the protein is denatured. Q115A is catalytically inactive under normal buffer conditions, in part due to a diminished affinity towards DNA. At low ionic strength and alkaline pH, as well as in the presence of Mn2+, i.e. under conditions where wild-type EcoRI shows a relaxed specificity, Q115A is active, however not as much as wild-type EcoRI. Under these conditions it cleaves the canonical sequence -GAATTC- with the same kcat/Km value as the sequence -GAAUTC-, which differs from the former sequence by a single methyl group, while wild-type EcoRI shows a tenfold lower kcat/Km for cleavage of -GAAUTC- than for -GAATTC-. Binding experiments, carried out in the absence of Mg2+, demonstrate that Q115A has a similar affinity towards -GAATTC- as to -GAAUTC-, while wild-type EcoRI binds to -GAATTC- with a tenfold preference over -GAAUTC-. On the basis of these thermodynamic and kinetic results it can be concluded that the hydrophobic contact between the γ-methylene group of Gln115 and the methyl group of the inner thymidine contributes about 3 kJ/mol (0.7 kcal/mol) to the energy of interaction, both in the ground and the transition state. Q115E is catalytically inactive under normal buffer conditions, but becomes active at low ionic strength or in the presence of Mn2+. Different from Q115A, Q115E is inactive at alkaline pH and its DNA binding affinity is highest at acidic pH. The dependence of its DNA cleavage activity on pH, which is governed by a pKa of 7.35, can be attributed to the protonation of the newly introduced glutamic acid residue, if it is assumed that the carboxyl group is located in a non-polar environment and/or involved in a hydrogen bond as the donor. Taken together, these results demonstrate that Gln115, as suggested on the basis of the revised EcoRI-DNA co-crystal structure, interacts with the inner thymidine of the EcoRI recognition sequence and has a structure stabilizing role. In addition, our results suggest that Gln115 is crucial for coupling specific DNA binding to catalysis under normal buffer conditions and we suggest that this coupling is achieved because direct and indirect involvement of Gln115 in base recognition induces local conformational alterations of the C-terminal region of β-strand β3, which contains Lys113 and Glu111 that are constituents of the catalytic centre of EcoRI. Activation of the catalytic centre, therefore, could be triggered by Gln115.
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页码:221 / 234
页数:14
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