Enzimas de restriccion

http://en.wikibooks.org/wiki/Structural_Biochemistry/Enzyme_Catalytic_Mechanism/Restriction_EnzymeStructural Biochemistry/Enzyme Catalytic Mechanism/Restriction EnzymeGeneral informationRestriction enzymes (also called restriction endonucleases) recognizes andcleaves DNA at specific sequences. Found in a wide range of bacterial species,these enzymes are valuable tools in the field of bioengineering as they are naturesscapple. Restriction enzymes serve the purpose of defending bacteria from viruswhere it degrades and digest foreign DNA. Restriction enzymes can also be usedfor cloning DNA fragments in the field of bioinformatics through the process ofBacterial Artificial Chromosomes (BAC) and Yeast Artificial Chromosomes (YAC).One example of such restriction enzyme is BamHI. This process is of highspecificity and uses restriction endonucleases to degrade the viral DNA. Specificbase sequences, recognition sites, are recognized by these enzymes. Theseenzymes usually recognize some specific sequence of DNA which is knownas recognition sites for the enzyme and cleave the DNA at certain positions.There are three different types of restriction enzymes: Types I, II and III. Types Iand III are large, multi-subunit complexes containing both the endonuclease andmethylase activities. Type I restriction enzymes cleave DNA at sites that can be1,000 base pairs away from the recognition site. Type III cleaves DNA at sitesabout 25 base pairs from the recognition sites. The most commonly used type isthe type II restriction enzyme. This type of restriction enzyme cleaves the DNA atthe site of recognition. 1

Each restriction enzyme cleaves DNA in a specific way. Some cuts the two DNAstrands asymmetrically, leaving unpaired bases at each end. This type of unpairedstrands of DNA is called a sticky end. Sticky ends of different but complementaryDNA strands can ligate to form a recombinant DNA. Restriction enzymes thatcleave directly at the opposing phosphodiester bond, leaving no unpaired bases onthe ends, are called blunt ends.The most widely studied restriction endonuclease is called "BamHI." Thisrestriction endonuclease provides an excellent example of how mostendonucleases behave in order to perform their assigned tasks. Like many otherrestriction endonucleases, BamHI binds to two divalent metals (such as Ca2+ orMg2+) to form a coordination compound that will increase its binding ability to DNA.However, instead of "searching" the entire DNA double-helix for the specificsequence it requires, BamHI uses a cellular shortcut: it latches onto the DNAmolecule at any convenient point, and then begins to slide along the DNAbackbone until it finds the sequence of interest. This behavior is not unique toBamHI; restriction endonucleases routinely perform such "tricks" in order to dotheir jobs more efficiently. This tactic is quite effective: instead of having to sortthrough the messy twists and turns of an entire DNA double-helix, the restrictionendonucleases only need to slide along one strand until they reach their targetsequence. They effectively turn a 3-D problem (searching through the bends andfolds of the DNA molecule) into a 2-D problem (sliding one way along a DNAstrand). For instance, restriction enzymes may choose to methylated nucleotidesequences that are not intended to be displaced through the addition of a methylgroup. This methylation marks the DNA sequence that will not be cut out from theoriginal sequence which could disrupt the whole cell. These methylated nucleotidesare formed as tags for the restriction enzymes to identify.Mechanisms for Cleavage by In-Line DisplacementRestriction enzymes catalyze the hydrolysis of the of the phosphodiester bonds inthe backbone of DNA. It specifically hydrolyzes the bond between 3’ oxygen andphosphorus to cause the bond to break. Resulting products are a DNA strand that 2

has a free 3’ hydroxyl group and another strand with a 5’ phosphoryl group. Thereare two methods that restriction enzymes can use to cleave the phosphodiesterbond in the DNA back bone. One way is to form covalent intermediates that can donucleophilic attacks to the Phosphate group at the phosphodiester linkages. Theother way is to simply hydrolyze the complex by adding water. The two methodsare shown below.Mechanism type 1 (Covalent intermediate)Nucleophile attacks the phosphoryl group and forms an intermediate. Then theintermediate is hydrolyzed and produces two separate DNA strands.Stereochemistry is retained in this mechanism - in the first step it is inverted butalso in the second step. Therefore, it retains the original stereo chemistry of thephosphorous atom.Mechanism type 2 (Direct Hydrolysis)An activated water molecule attacks the phosphorus atom directly. There is nointermediate step in this kind of mechanism, such reactions are known asconcerted reactions. With this kind of mechanism, the stereochemistry of moleculeis inverted. This mechanism is almost identical to the second order nucleophilic 3

substitution (SN2) reaction. The attack of water on the phosphorus atom is drivenby the fact that the electronegative oxygen, equipped with 2 lone pairs of electrons,is attracted to the phosphorus which is relatively electropositive because much ofits electron density is drawn away by the oxygen containing groups attached to it.The reason the reverse reaction (the R1OH alcohol product attacking thephosphorus and kicking off the water) does not take place is first, the alcohol is abit more bulky and will thus encounter more steric hindrance from the groupsattached to the phosphorus and second, and more importantly, this reactionusually takes place in an aqueous solvent where the is a lot of water. Thus,according to LeChatliers Principle, the excess of one of the reactant of the reactionshown below will push the equilibrium towards the products.MagnesiumEnzymes acting on phosphate containing substrates usually require divalentcations such as magnesium for activity. There are several ways to determine thepresence of metal ions. One way is to prepare crystals of EcoRV bound tooligonucleotides that contain the enzymes recognition sequence without themagnesium to prevent cleavage. After the crystals have been formed, they aresoaked in a metal ion solution. Another way to prepare the crystals is to use amutated form of an enzyme that is less active. In this way, no cleavage takes placeso binding sites can be determined since it is impossible to view the complex inEcoRV and cognated DNA molecules with magnesium present as the enzymescleaves the substrate under this condition. A maximum of three metal ions can befound in each active site. The metal ions are coordinated to the protein through twoaspartate residues and to one of the phosphoryl group oxygen atoms near thecleavage site which helps water attack the phosphorous atom like the zinc incarbonic anhydrase. 4

SpecificityMost restriction enzymes have recognition sequences that are inverted repeatsthat gives a 3D structure of the recognition site a twofold rotational symmetry. Thesymmetry between the recognition sequence and the enzyme facilitates therecognition of cognate DNA. The binding affinity for substrates can help todetermine specificity as well. The enzyme only cleaves cognate sequencesbecause within the GATATC sequence, the G and A bases at the end of the 5 endof each strand and their base partners are in direct contact with the enzymethrough hydrogen bonds with residues located on the loops. This sequence has akink in the center due to the TA base pairs at the center allowing for the distortionof DNA. Noncognate sequences, on the other hand, are not distorted enough.Thus, the lack of distortion means that there is no phosphate positioned closeenough to the active site aspartate residues to complete a magnesium binding siteso nonspecific complexes are unable to bind magnesium ions and complete thecatalytic apparatus. Distortion of the substrate and magnesium binding are themain factors that contribute to catalytic specificity. Distorted DNA increases bindingenergy since it makes additional contacts with the enzyme but it is cancelled out bythe energy it takes to distort the DNA. In EcoRV this causes a slight difference inbinding affinity between cognate and nonspecific DNA but it cognate complexes, itaffects catalysis through the completion of magnesium binding. Distortion alsohelps protect host-cell DNA by blocking catalysis through methylation since thepresence of methyl groups block the formation of hydrogen bonds between theamino group of an adenine nucleotide of the recognition site and the side chain ofthe asparagine residue that is closely linked to the DNA.Restriction enzymes bind indiscriminately to any DNA sequence but only specificnon-methylated cognate sequences are cleaved.Take EcoRV endonuclease for example,the enzyme recognises the sequence5–GATATC–33–CTATAG–5and cuts specifically at the center. 5

This recognition site has special features: It is made of inverted repeats, known aspalindromic sequences; and it is symmetric around axis of rotation, giving a 2-foldrotational symmetry.The dimeric enzyme matches this 2-fold symmetry of the substrate DNA andsurrounds the DNA in tight embrace. On binding with the DNA, the enzyme formshydrogen-bonds to the G and A bases at the 5 end of each strand and theircorresponding Watson-Crick partners. The central TA base pairs do not formhydrogen bonds but are essential to the distortion of the DNA recognition site asthey are most easily deformed.The Eco RI restriction enzymeAn example of post-reactive cognate DNA-Eco RI complex at 2.5A in the presenceof Mn2+ ion. 6

Although the additional interactions made by the distorted cognate DNA withEcoRV have led to an increase in its binding energy, this additional energy iscanceled as the DNA distorts itself from the relaxed conformation. Therefore, bothcognate and non-cognate complexes have about the same free energy in the end,accounting for the little difference in their binding affinity. In the non-cognatecomplex, however, DNA phosphates are too far away from the Asp residues tomake up a Mg-binding site. Thus, the DNA will be bound but not cleaved.BamHIBamHI is the most studied restriction endonuclease. It is derived from Bacillusamyloliquefaciens. GGATCC is its recognition site, and leaves a sticky end. Its adimer and each monomer binds to its recognition site. Persistent issues with thisspecific enzyme spontaneously failing to generate cloneable fragments necessitatealiquoting prior to use. Questions such as How does BamHI cleave DNA? Howcan BamHI find its specific site among the thousands of nonspecific sites? Dr.Hector co-crystallied the complex BamHI DNA with divalent cations(missing somees) such as Ca2+ and Mn2+. Ca2+ binds to active site but reaction does not carry 7

out catalysis. Its catalytic mechanism consist of pre-reactive state, transition state,post-reactive state. Dr. Hector also crystallized BamHI with a non-specific DNA.DNA is far from active site of enzyme. Restriction endonuclease first binds to anyDNA and until find right side they cleave it. Three main thermodynamic propertiesof a protein/non-specific DNA complex: 1. More hydrated at teh protein DNAinterface. 2. Stabilized primarily by electrostatic interactions. 3. Experience low-heat capacity change upon binding.Mechanism: The mechanism that BamHI uses is essentially the Sn2 mechanism.The amino acid E113 activates a water molecule by drawing a proton away from itand making the water oxygen atom more like a hydroxide oxygen atom. Theactivated water attacks the phosphorous atom of the phosphodiester bond that isto be broken. The transition state that forms is trigonal bipyrimadal and carries anadditional negative charge that is stabilized by the two divalent metal ions. Theleaving group is the alkoxide attached to the C3 carbon. A second water moleculedonates a proton to neutralize the alkoxide and form the free 3 end of the DNAstrand. The proton that the first water molecule lost to E113 can be proton shuttledto the hydroxide ion formed when the second water molecule donated its protonand the enzyme is back to where it started. The final result is a strand with a free 3end and a phosphorylated 5 end.Type II Restriction EnzymesType II restriction enzymes have a structure containing beta strands with aspartateresidues forming the magnesium binding sites present in Archaea and Bacteria.The sequences show that bacteria most likely obtained the genes encoding theenzymes from other species through horizontal gene transfer which is the passingof DNA fragments that provide selective advantage in a certain environmentbetween different species. 8

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Enzimas de restriccion

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Enzimas de restriccion — Document Transcript