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From Proteopedia

Revision as of 04:24, 4 April 2012

YourMacromolecule

Introduction

EcoRV endonuclease is a type II restriction enzyme found in e. coli bacteria. Restriction enzymes recognize and cleave specific sequences of DNA. EcoRV cleaves the AAAGATATCTT. A type II restriction enzymes cleave at a short distance from the recognition site and often use Mg(2+) as a cofactor, as does this enzyme.

Outline -found in e. coli -is a type II restriction endonuclease/enzyme -explain restriction enzymes---cut DNA -explain differences between type II and others -works in presence of Mg(2+) -cuts specific section of DNA

Overall Structure

EcoRV endonuclease is functional as a dimer consisting of two monomers; both monomers depicted are shown in green or purple in a U shape. Each monomer, shown , consists of 244 amino acids, 9 alpha helices, shown , in pink and 11 beta strands, shown , in blue. The beta strands form 3 beta sheets of various sizes, both parallel and anti parallel. The is a mixture of parallel and anti-parallel strands. The light purple and dark purple strands are two anti-parallel sheets. The connection between the dark and light purple beta sheets is parallel

Binding Interactions

DNA recognition sites on the EcoRV molecule, called R-loops, bind to the major grooves of the double stranded DNA at its recognition sequence GATATC by hydrogen bonding. This enzyme is a type II restriction endonuclease, which means this enzyme cleaves the DNA recognition sequence at the center (between the T and A base pairs). These hydrogen bonds makes the DNA form a kinked conformation that is later stabilized by the addition of the Mg2+ ion. The Mg2+ ion is a catalyst that causes the DNA to shift in a way that increases the rate necessary for DNA cleavage.

The Mg2+ binding site is formed when ionic interactions cause the slightly acidic Asp90 residue and the slightly negatively charged scissile phosphodiester group to approach each other. This allows the Mg2+ ion to bind to this enzyme, also with ionic interactions between the positively charged Mg2+ and the partially negative charged oxygen atoms from multiple molecules. These molecules that bind to the Mg2+ ion are the carboxylate oxygen atoms from the Asp74 and Asp90 residues, the nonesterified oxygen from the scissile phosphodiester group, and three additional oxygen atoms from three water molecules. These six ionic bonds form an octahedral shape in the active site of this enzyme.

These six ionic interactions all have about the same binding distance except for one bond between the oxygen from the Asp74 residue and the Mg2+ ion that is significantly longer. The five similar bond lengths are all about 2.08 Å, but the bond between Mg2+ and the Asp74 oxygen spans a distance of 2.9 Å. This is noted because the Asp90 and scissile phosphodiester molecules that bind to this Mg2+ ion change their bonding interactions with hydrogen to accommodate the addition of the Mg2+ ion. The Asp74 residue maintains its hydrogen bond interactions on its side chain with the main chain of the Ile91 residue and the water molecule, which is why it keeps a greater distance between itself and the Mg2+ ion.

Additional Features

Credits

Introduction - Jesse

Overall Structure - Nicole

Drug Binding Site - Julia

Additional Features - Sam

References