CH2216 - Enzymes and Nucleic Acids

8/9/2019 CH2216 - Enzymes and Nucleic Acids

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CH2216 Chemical Biology

The human body contains a vast array of chemical processes, and a large range of chemical species

to perform them.

77% of the body is made of

water, Ions inorganic species such as Na+ H2PO4-and K+ and small molecules amino acids, sugars, nucleotides, fatty acids etc

The remaining 23% consists of macromolecules, such as nucleic acids (DNA) and proteins.

Most of the chemical processes that occur within the body, in particular those that form vital

components require a catalyst to occur. These catalysts are present to ensure that energetically

unfavourable gaps can be breached and to prevent the overproduction of unwanted and toxic side

products through stereospecificity. These catalysts within the cells usually take the form of proteins

(as enzymes) or as RNA (in ribosomes and ribozymes), though most are proteins.

The catalyst itself is a description of the probability that compound A will be converted into

compound B, essentially it offers a lower energy route over which a reaction can occur, increasing

the speed with which a reaction can reach completion.

For example the conversion of glucose to glucose-6-phosphate and the release of a phosphate group

from ATP to form ADP;


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Proteins are made of amino acids, a group of molecules for which the chemical and physical

properties are well documented, it is, however, impossible to determine the behaviours of a protein

based upon its amino acid units. The sequence of amino acids is responsible for the properties of a

protein, and therefore its ability to act as a catalyst. There are 20 proteinogenic amino acids and

although it is possible to determine the amino acid sequence by the sequencing of the

corresponding gene the properties of the protein still cannot be predicted.

Amino acids get their name from the two functional groups present in each, the amino and

carboxylic groups, the third group always being hydrogen. The remaining functionality on the carbon

centre can be anything and is often labelled R. This variation inherent to each amino acid means

that, unless R is to be a hydrogen atom (as in glycine), each amino acid will display asymmetry and in

turn stereochemistry, being able to form one of (at least) two enantiomers. This is the basis of

classification of amino acids and it is important to note that each enantiomer will behave differently

biologically.

Fischer-Projections

-Orient carbon chain vertically.

-Put carbon atom with highest oxidation number on top (usually the one carrying most oxygen

atoms).

-Make the asymmetric carbon atom pointing towards you, the 2 next C-neighbours away from you.

-Decide whether the amino group (or other relevant group) is pointing to the left or to the right side.


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The Amino Acids

One important trait of an amino acid; Cysteine is redox active;


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There is also, in fact, a 21st

amino acid, selenocysteine;

Amino acids can take on several forms, the carboxylic group can be deprotonated and the amino

group protonated depending on the pH of the environment in which the amino acid is found. At

neutral pH both the groups are altered to form the zwitterionic amino acid;

The dissociation of amino acids is related to the pKa (the tendency towards deprotonation) which in

turn is related to pH through the Henderson-Hasselbach equation;

pH = pKa + log[A]

[HA]

The pKa of any acid is equal to the pH at which half of the molecules are dissociated and half are

neutral.

Biosynthesis of Proteins

In the synthesis of proteins only L-enantiomers and -amino acids (the amino group is attached to

the carbon in the carboxylic group by peptide bonds) are used. A protein is formed of un-branched

single chains of amino acid monomers. The three-dimensional structure of the protein will be

determined by the sequence of amino-acids that it comprises of and in turn this structure

determines the functionality of the protein.


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There are four levels of structure in proteins;

1. Primary the linear sequence of amino acids2. Secondarythe localised organisation of parts of a polypeptide chain (i.e. -helices or -

sheets)

3. Tertiary the overall three-dimensional arrangement of the polypeptide chain4. Quaternary the association of two or more polypeptide is into a multi-subunit complex

A regular combination of secondary structures is called a motif, there are several common motifs. A

coiled coil motif is formed by two or more -helices coiled around each other, the helix-loop-helix

motif is also commonly seen, as is the zinc finger motif.

A newly synthesised polypeptide chain will undergo folding and chemical modification in order to

generate the final protein, all protein species adopt a single conformation (the native state) which isthe most stable folded form of the molecule. The information for protein folding is encoded in the

sequence, if a protein is unfolded it will, given the right conditions, refold into the same folded form.

Within living organisms this folding is promoted by chaperones.

The biological activity of proteins can be altered chemically and many chemically modified proteins

are important biological agents, i.e. -carboxyglutamate found in pro-thrombin an essential blood

clotting factor.

An important post-translational modification of proteins is the ubiquitin-mediated degradation

pathway. Ubiquitin is a small, highly-conserved regulatory protein that is ubiquitously expressed in

eukaryotes. Ubiquitination is the covalent addition, via isopeptide bond, of one or more ubiquitin


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monomers beginning with the activiation of ubiquitin via an E1 enzyme. The overall action is to

break a protein into its peptide monomers.

An aberrantly folded protein is often the cause of slow developing diseases, for example amyloid

plaques in Alzheimers is formed by a tangle of protein filaments.

The function of a protein often involves a conformational change; proteins are designed to bind to a

range of molecules. This binding is characterised by two properties; affinity and specificity. Anti-

bodies exhibit precise ligand-binding specificity, enzymes are highly efficient and specific catalysts.

There are also mechanisms in place to prevent protein activity or regulate it, four of the most

common are;

Allosteric transitions, the release of catalytic subunits to induce the active state of anenzyme

Phosphorylation/dephosphorylation

Proteolytic activiation, the breaking of an enzyme into smaller chains to produce the activeprotein, i.e chymotripsinogen to the active -chymotripsin form

Compartmentalisation


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Enzyme Kinetics

A basic plot of rate against substrate concentration can be annotated in a particular way;

These values are linked by the Michaelis-Menten equation;

V0 =Vmax [S]

Km + [S]

If there is a low [S] value V0 shows a linear dependence, if [S] is much larger than Km then V0 will

plateau at Vmax (i.e. V0=Vmax).

In an enzymatic reaction if the concentration of enzyme, [E], is doubled, Vmax will also double,

however Km will remain the same. Km will only change if the affinity of the substrate is changed, a low

affinity substrate will approach the same value of Vmax at a slower rate. Km values for several

enzymes;


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The turnover number of an enzyme is defined as Kcat, this is the number of substrate units an

enzyme can convert per second. Some enzymes, such as catalase for the enzymatic conversion of

hydrogen peroxide, can achieve Kcatvalues of 40,000,000 units per second however that isnt to say

that is an achievable turnover within the body. There is an imposed diffusion-controlled limit of 108-

109M

-1s

-1, Kcat/Km will give values in the correct units for comparison and those of several common

enzymes are given in the table below;

It is possible to get the Michaelis-Menten equation into the form y=mx+c to form a Lineweaver-Burk

plot, this can then be used to calculate various values in the Michaelis-Menten equation in cases

where they arent already known.

maxmax0

maxmax0

max0

max

0

1

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1

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][1

][

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VSV

K

V

SV

S

SV

K

V

SV

SK

V

SK

SVV

m

m

m

m


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There are different types of enzymatic reaction, some involve the formation of a ternary complex

and some dont, these can be shown as;

By plotting 1/V0(1/M/min) against 1/*S1](1/mM) while increasing the concentration of S2 it can be

determined whether a ternary complex is formed or not.

The first image represents a reaction in which a ternary complex (ES1S2) is formed, the second

represents a reaction which undergoes via double displacement.

Enzyme Inhibition

Enzymes are susceptible to both reversible and irreversible forms of inhibition. An enzyme inhibitor

is almost always a small molecular agent that interferes with catalysis, some are naturally occurring

regulators of enzymatic processes but some are artificial agents such as aspirin (which inhibits thefirst enzyme in the prostaglandin pathway).

Reversible Inhibition

-Competitive inhibition


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The rate of a competitively inhibited reaction can be equated as;

Where KI is the dissociation constant for the

enzyme/inhibitor complex and Km is the

apparent Km.

-Uncompetitive inhibition

Where;

Where KI is the dissociation constant for the

inhibitor/substrate complex.

-Mixed inhibition

][

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0

EI

IEK

K

I

SK

SVV

I

I

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ESI

IESK

K

I

SK

SVV

I

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m


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Where;

These different types of inhibition will affect the values of Vmax and apparent Km differently;

Irreversible Inhibition

Irreversible inhibitors bind covalently to the enzyme or destroy a functional group necessary for

catalysis. There is a special class of irreversible inhibitors called suicide inhibitors, or mechanism

based inhibitors, these are relatively non-reactive until they bind to the active site of a specific

enzyme. They undergo the first few mechanistic steps as usual before generating a very reactive

compound that reacts irreversibly with the enzyme, essentially the enzyme creates its own inhibitor

(i.e. -lactam inhibitors, penicillin etc.).

An example of irreversible inhibition is the action of diisopropylfluorophosphate on chymotripsin.

Chymotripsin is a protease the catalyses the hydrolytic cleavage of peptide bonds, it is specific for

peptide bonds adjacent to aromatic amino acid side chains (Phe, Tyr and Trp) enhancing the rate of

hydrolysis by a factor of 109.

DIFP covalently binds to the serine within chymotripsin

preventing the action of the enzyme.

]['

][max

0SK

SVV

m


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Chymotripsin in Detail


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Catalysis of Biochemical Reactions by Proton Transfer

Many biochemical reactions involve the formation of unstable charged intermediates that tend to

break down rapidly into the original reactants, the charge on these intermediates can be stabilisedby the transfer of a proton to form a new species that will break down rapidly to form the desired

products. Catalysis of this type using only water (as H2O, H+

or

OH) is known as specific acid-base

catalysis, if the proton transfer is mediated by other classes of molecule (e.g. organic acids or bases)

it is referred to as general acid-base catalysis.

Covalent Catalysis

In covalent catalysis a bond is formed between the enzyme (with a nucleophilic group, X:) and the

substrate;

A-BA + B (hydrolysis by water, no enzyme)

A-B + X: A-X + BA + X: + B

The nucleophile could be in the form of an amino acid side-chain or a enzyme cofactor.

Hexokinase

Hexokinase is used in the phosphorylation of-D-glucose at the 6 position by Mg.ATP, it undergoes

an induced fit mechanism. The O-H group at C6 is similar in reactivity to water, and water is able to


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enter the active site of the enzyme however hexokinase favours the reaction with glucose by a factor

of 106. This discrimination is due to a conformational change when the correct substrate binds, in

the absence of glucose the enzyme is in an inactive conformation with the active site amino acid side

chains in the wrong position to perform the reaction. When a molecule of glucose binds the binding

energy involved induces a conformational change bringing the amino acid side chains into the

correct position, they then participate in general acid-base catalysis and transition state stabilisation.

This process prevents the hydrolysis of ATP with water.

This selectivity can be bypassed by another sugar, xylose, a 5-carbon sugar that is stereochemically

similar to glucose and will also bind to hexokinase, but not in a position that allows it to be

phosphorylated. Regardless xylose does increase the rate of hydrolysis of ATP, the binding of xylose

is sufficient to induce a conformational change to the active conformation, tricking the enzyme into

phosphorylating water.

Enolase

Enolase is used in the conversion of 2-phosphoglycerate to phosphoenolpyruvate, a lysine residue

initiate the reaction as a general base catalysis to form an enolic intermediate while Mg2+

ions

stabilise the negative charge on the oxygen atoms of the carboxyl group, a glutamate residue thencompletes the reaction by general acid catalysis to form and release the product.

Pyruvate Decarboxylase and Thiamine Pyrophosphate

Thiamine pyrophosphate is derived from thiamine, vitamin B1, a lack of which leads to a conditionknown as beriberi (accumulation of body fluids, swelling, eventually leading to death). TPP plays an

important role in the cleavage of bonds adjacent to carbonyl groups (e.g. the decarboxylation of-

keto acids) and in chemical rearrangements in which an activated acetaldehyde is transferred from

one carbon to another. The functional part of TPP is a thiazolium ring with a highly acidic proton.


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Pyruvate decarboxylase, aided by TPP, is involved in the conversion of pyruvate to acetaldehyde, and

consequently ethanol.

1-Nucleophilic attack of the TPP carbanion

2-Decarboxylation produces a carbanion that is resonance

stabilised by the thiazolium ring (acts as an electron sink)

3-Protonation yields hydroxyethyl TPP

4-Release of Aldehyde

5-Proton dissociates to reform the anion

The electron sink effect of the thiazolium ring is due to

the electron deficient structure formed into which the

carbanion electrons can be delocalised by resonance.

The acetaldehyde is then converted into ethanol by alcohol dehydrogenase and NADH, with a Zn

2+

stabilising cation.

Recombinant DNA

DNA is cloned using plasmid vectors, recombinant DNA technology is dependent on the ability to

produce large numbers of identical DNA molecules. This is typically achieved by placing a DNA

fragment of interest into a vector DNA molecule which can replicate within a host cell, when a single


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DNA fragment is introduced into a host cell a large number of the fragment is reproduced along with

the vector. Two common vectors used are E.coliplasmid vectors and bacteriophage vectors.

A plasmid is an extrachromosomal self-replicating DNA molecule;

The desired DNA fragment is enzymatically inserted into the plasmid vector to form a recombinant

plasmid. This is then mixed with E. colicells in presence of CaCl2 on a culture of nutrient agar plates

containing ampicillin, this kills the E. colicells that do not take up the plasmid and leaves only the

cells which have taken up the plasmid vector. These cells then replicate naturally, producing a large

number ofE. colicells containing the plasmid ready for extraction. Plasmid cloning permits the

isolation of DNA fragments from complex mixtures.

To extract the DNA fragments desired a restriction enzyme is used, these cut DNA molecules at

specific sequences.


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If necessary a molecule such as EcoRI methylase can methylate the sequence in specific places

preventing the action ofEcoRI which will not cleave methylated DNA. By selecting the correct

restriction enzymes the desired, reproducible DNA fragment can be properly extracted.


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DNA molecules can be resolved using gel electrophoresis, the mixture of fragments is placed into a

well of agarose or polyacrylamide gel, to which an electric field is applied. The molecules will move

through the pores in gel at a rate inversely proportional to their chain length, if the fragments are

incubated with a fluorescent dye this separation can be visualised.

Polymerase Chain Reactions

Polymerase chain reactions are an alternative to cloning, it can be used to amplify specific DNA

sequences from a complex mixture when the ends of the sequences are known. PCR amplification of

mutant alleles allows the detection of human genetic disease, PCR is also used to amplify DNA

sequences for cloning, as probes and for use in forensics.


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Modifications and mutations can also be introduced using PCR, in

this example EcoRI sites are created at both ends of the DNA

fragment.

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CH2216 - Enzymes and Nucleic Acids