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http://en.wikibooks.org/wiki/Structural_Biochemistry/Enzyme_Catalytic_Mechanism/Restriction_

Enzyme

Structural Biochemistry/

Enzyme Catalytic Mechanism/

Restriction Enzyme

General information

Restriction enzymes (also called restriction endonucleases) recognizes and

cleaves 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 nature's

scapple. Restriction enzymes serve the purpose of defending bacteria from virus

where it degrades and digest foreign DNA. Restriction enzymes can also be used

for cloning DNA fragments in the field of bioinformatics through the process of

Bacterial Artificial Chromosomes (BAC) and Yeast Artificial Chromosomes (YAC).

One example of such restriction enzyme is BamHI. This process is of high

specificity and uses restriction endonucleases to degrade the viral DNA. Specific

base sequences, recognition sites, are recognized by these enzymes. These

enzymes usually recognize some specific sequence of DNA which is known

as 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 I

and III are large, multi-subunit complexes containing both the endonuclease and

methylase activities. Type I restriction enzymes cleave DNA at sites that can be

1,000 base pairs away from the recognition site. Type III cleaves DNA at sites

about 25 base pairs from the recognition sites. The most commonly used type is

the type II restriction enzyme. This type of restriction enzyme cleaves the DNA at

the site of recognition.

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Each restriction enzyme cleaves DNA in a specific way. Some cuts the two DNA

strands asymmetrically, leaving unpaired bases at each end. This type of unpaired

strands of DNA is called a sticky end. Sticky ends of different but complementary

DNA strands can ligate to form a recombinant DNA. Restriction enzymes that

cleave directly at the opposing phosphodiester bond, leaving no unpaired bases on

the ends, are called blunt ends.

The most widely studied restriction endonuclease is called "BamHI." This

restriction endonuclease provides an excellent example of how most

endonucleases behave in order to perform their assigned tasks. Like many other

restriction endonucleases, BamHI binds to two divalent metals (such as Ca2+ or

Mg2+) to form a coordination compound that will increase its binding ability to DNA.

However, instead of "searching" the entire DNA double-helix for the specific

sequence it requires, BamHI uses a cellular shortcut: it latches onto the DNA

molecule at any convenient point, and then begins to slide along the DNA

backbone until it finds the sequence of interest. This behavior is not unique to

BamHI; restriction endonucleases routinely perform such "tricks" in order to do

their jobs more efficiently. This tactic is quite effective: instead of having to sort

through the messy twists and turns of an entire DNA double-helix, the restriction

endonucleases only need to slide along one strand until they reach their target

sequence. They effectively turn a 3-D problem (searching through the bends and

folds of the DNA molecule) into a 2-D problem (sliding one way along a DNA

strand). For instance, restriction enzymes may choose to methylated nucleotide

sequences that are not intended to be displaced through the addition of a methyl

group. This methylation marks the DNA sequence that will not be cut out from the

original sequence which could disrupt the whole cell. These methylated nucleotides

are formed as tags for the restriction enzymes to identify.

Mechanisms for Cleavage by In-Line Displacement

Restriction enzymes catalyze the hydrolysis of the of the phosphodiester bonds in

the backbone of DNA. It specifically hydrolyzes the bond between 3’ oxygen and

phosphorus to cause the bond to break. Resulting products are a DNA strand that

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has a free 3’ hydroxyl group and another strand with a 5’ phosphoryl group. There

are two methods that restriction enzymes can use to cleave the phosphodiester

bond in the DNA back bone. One way is to form covalent intermediates that can do

nucleophilic attacks to the Phosphate group at the phosphodiester linkages. The

other way is to simply hydrolyze the complex by adding water. The two methods

are shown below.

Mechanism type 1 (Covalent intermediate)

Nucleophile attacks the phosphoryl group and forms an intermediate. Then the

intermediate is hydrolyzed and produces two separate DNA strands.

Stereochemistry is retained in this mechanism - in the first step it is inverted but

also in the second step. Therefore, it retains the original stereo chemistry of the

phosphorous atom.

Mechanism type 2 (Direct Hydrolysis)

An activated water molecule attacks the phosphorus atom directly. There is no

intermediate step in this kind of mechanism, such reactions are known as

concerted reactions. With this kind of mechanism, the stereochemistry of molecule

is inverted. This mechanism is almost identical to the second order nucleophilic

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substitution (SN2) reaction. The attack of water on the phosphorus atom is driven

by the fact that the electronegative oxygen, equipped with 2 lone pairs of electrons,

is attracted to the phosphorus which is relatively electropositive because much of

its electron density is drawn away by the oxygen containing groups attached to it.

The reason the reverse reaction (the R1OH alcohol product attacking the

phosphorus and kicking off the water) does not take place is first, the alcohol is a

bit more bulky and will thus encounter more steric hindrance from the groups

attached to the phosphorus and second, and more importantly, this reaction

usually takes place in an aqueous solvent where the is a lot of water. Thus,

according to LeChatlier's Principle, the excess of one of the reactant of the reaction

shown below will push the equilibrium towards the products.

Magnesium

Enzymes acting on phosphate containing substrates usually require divalent

cations such as magnesium for activity. There are several ways to determine the

presence of metal ions. One way is to prepare crystals of EcoRV bound to

oligonucleotides that contain the enzyme's recognition sequence without the

magnesium to prevent cleavage. After the crystals have been formed, they are

soaked in a metal ion solution. Another way to prepare the crystals is to use a

mutated form of an enzyme that is less active. In this way, no cleavage takes place

so binding sites can be determined since it is impossible to view the complex in

EcoRV and cognated DNA molecules with magnesium present as the enzymes

cleaves the substrate under this condition. A maximum of three metal ions can be

found in each active site. The metal ions are coordinated to the protein through two

aspartate residues and to one of the phosphoryl group oxygen atoms near the

cleavage site which helps water attack the phosphorous atom like the zinc in

carbonic anhydrase.

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Specificity

Most restriction enzymes have recognition sequences that are inverted repeats

that gives a 3D structure of the recognition site a twofold rotational symmetry. The

symmetry between the recognition sequence and the enzyme facilitates the

recognition of cognate DNA. The binding affinity for substrates can help to

determine specificity as well. The enzyme only cleaves cognate sequences

because within the GATATC sequence, the G and A bases at the end of the 5 end

of each strand and their base partners are in direct contact with the enzyme

through hydrogen bonds with residues located on the loops. This sequence has a

kink in the center due to the TA base pairs at the center allowing for the distortion

of DNA. Noncognate sequences, on the other hand, are not distorted enough.

Thus, the lack of distortion means that there is no phosphate positioned close

enough to the active site aspartate residues to complete a magnesium binding site

so nonspecific complexes are unable to bind magnesium ions and complete the

catalytic apparatus. Distortion of the substrate and magnesium binding are the

main factors that contribute to catalytic specificity. Distorted DNA increases binding

energy since it makes additional contacts with the enzyme but it is cancelled out by

the energy it takes to distort the DNA. In EcoRV this causes a slight difference in

binding affinity between cognate and nonspecific DNA but it cognate complexes, it

affects catalysis through the completion of magnesium binding. Distortion also

helps protect host-cell DNA by blocking catalysis through methylation since the

presence of methyl groups block the formation of hydrogen bonds between the

amino group of an adenine nucleotide of the recognition site and the side chain of

the asparagine residue that is closely linked to the DNA.

Restriction enzymes bind indiscriminately to any DNA sequence but only specific

non-methylated cognate sequences are cleaved.

Take EcoRV endonuclease for example,

the enzyme recognises the sequence

5'–GATATC–3'

3'–CTATAG–5'

and cuts specifically at the center.

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This recognition site has special features: It is made of inverted repeats, known as

palindromic sequences; and it is symmetric around axis of rotation, giving a 2-fold

rotational symmetry.

The dimeric enzyme matches this 2-fold symmetry of the substrate DNA and

surrounds the DNA in tight embrace. On binding with the DNA, the enzyme forms

hydrogen-bonds to the G and A bases at the 5' end of each strand and their

corresponding Watson-Crick partners. The central TA base pairs do not form

hydrogen bonds but are essential to the distortion of the DNA recognition site as

they are most easily deformed.

The Eco RI restriction enzyme

An example of post-reactive cognate DNA-Eco RI complex at 2.5A in the presence

of Mn2+ ion.

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Although the additional interactions made by the distorted cognate DNA with

EcoRV have led to an increase in its binding energy, this additional energy is

canceled as the DNA distorts itself from the relaxed conformation. Therefore, both

cognate 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-cognate

complex, however, DNA phosphates are too far away from the Asp residues to

make up a Mg-binding site. Thus, the DNA will be bound but not cleaved.

BamHI

BamHI is the most studied restriction endonuclease. It is derived from Bacillus

amyloliquefaciens. G'GATCC is its recognition site, and leaves a sticky end. It's a

dimer and each monomer binds to its recognition site. Persistent issues with this

specific enzyme spontaneously failing to generate cloneable fragments necessitate

aliquoting prior to use. Questions such as 'How does BamHI cleave DNA? How

can BamHI find its specific site among the thousands of nonspecific sites? Dr.

Hector co-crystallied the complex BamHI DNA with divalent cations(missing some

e's) such as Ca2+ and Mn2+. Ca2+ binds to active site but reaction does not carry

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out catalysis. It's 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 any

DNA and until find right side they cleave it. Three main thermodynamic properties

of a protein/non-specific DNA complex: 1. More hydrated at teh protein DNA

interface. 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 it

and making the water oxygen atom more like a hydroxide oxygen atom. The

activated water attacks the phosphorous atom of the phosphodiester bond that is

to be broken. The transition state that forms is trigonal bipyrimadal and carries an

additional negative charge that is stabilized by the two divalent metal ions. The

leaving group is the alkoxide attached to the C3' carbon. A second water molecule

donates a proton to neutralize the alkoxide and form the free 3' end of the DNA

strand. The proton that the first water molecule lost to E113 can be proton shuttled

to the hydroxide ion formed when the second water molecule donated its proton

and the enzyme is back to where it started. The final result is a strand with a free 3'

end and a phosphorylated 5' end.

Type II Restriction Enzymes

Type II restriction enzymes have a structure containing beta strands with aspartate

residues forming the magnesium binding sites present in Archaea and Bacteria.

The sequences show that bacteria most likely obtained the genes encoding the

enzymes from other species through horizontal gene transfer which is the passing

of DNA fragments that provide selective advantage in a certain environment

between different species.