lecture 11 - introduction to enzymes

8/18/2019 Lecture 11 - Introduction to Enzymes

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Enzymes

Bacterial Restriction endonucleases


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What are enzymes?

• enzymes are biological catalysts

• usually proteins but RNA can also act as enzymes under specific conditions as well

• catalysts are compounds that increase the rate of reactions without beingconsumed in the reaction

• the rate of chemical reactions depend upon the concentrations of participants(reactants and products) and the activation energy required for a reaction tooccur

• in biological systems, the concentrations of reactants are usually very low and

significant activation energy barriers exist. Therefore, most biochemicalreactions would occur too slowly to permit life in the absence of catalysts.

• enzymes increase the rate of reactions by addressing low reactant concentrationsand high activation energies


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Enzymes do not alter reaction equilibria

“An enzyme cannot alter the laws of thermodynamics and consequently cannot alter the equilibrium of a chemical reaction” Stryer

consider the reaction of Substrate (S) to Product (P):

Note that in the enzyme catalyzed reaction, the rate of product formation levelsoff with time. This is because the reaction has reached equilibrium: substrate

is still being converted to product but product is also being converted into substrateat a rate such that the product remains constant.

S P Note that with or withoutenzyme present the sameamount of product willbe formed in a chemicalreaction.With enzyme the reactionmay take seconds andwithout may take hours(or seconds vs centuriesdepending on the reaction).


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S P

suppose in a chemical reaction that:the rate of product formation (k 1) = 1 x 10 -4 mol/sandthe rate of substrate formation (k -1) = 1 x 10 -6 mol/s

The equilibrium constant for the reaction is given by the ratio of the rate constants

in other words, when the reaction is at equilibrium the ratio of product to substrateis 100:1

the presence of an enzyme catalyzing the reaction will not alter this ratio (equilibrium).but it may reduce the time to reach this equilibrium from hours or centuries to secondsor a fraction of second

10 -4 s-1

10 -6 s-1

[S] K

[P]=

10 -6

10 -4= = 100


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many reactions take hours, years, or thousands of years to proceed to completion(equilibrium) but enzymes can increase rates millions of times


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orotidine monophosphateconversion touridine monophosphatebyOMP decarboxylase

half-life of OMP in absence of enzyme = 78 million yearshalf-life of OMP in presence of enzyme = 18 milliseconds


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"OMPDC Carbanion Mechanism" by Shareef164 - I Michael created this work entirely by myself.. Licensed under CC BY -SA 3.0 via Commons -https://commons.wikimedia.org/wiki/File:OMPDC_Carbanion_Mechanism.png#/media/File:OMPDC_Carbanion_Mechanism.png

OMP decarboxylase reaction mechanism

http://www.google.ca/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRxqFQoTCJG8oa2zxMgCFQRWPgodj2MDuQ&url=http://scop.berkeley.edu/sunid=51376&ver=1.75&psig=AFQjCNG5zoCLe1p33axiDgkZwm695ZPVGQ&ust=1444995881243449


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Enzymes increase the Rate of Reactions by decreasing activation energy

• many of the reactions that occur in cells will occur spontaneously (- ΔG) but occurtoo slowly for life processes

consider the oxidation of glucose

C6H12O6 + 6 O2 → 6 CO 2 + 6 H2O

the ΔG is about -2872 kJ/mol and a large amount of heat is liberated in thisspontaneous reaction.

• The equilibrium for this reaction is far on the right side of the above equationbecause thermodynamically, the products are less energetic and therefore more stableand less ordered.

• The presence of glucose is not favored in this universe (at least in the presence ofoxygen) yet a bottle of glucose can sit on a shelf for hundreds of years and not

become substantially oxidized.


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• An enzyme can quickly bring about the oxidation of glucose howeverby lowering the energy barrier to the forward (oxidation) reaction

• Note that a spontaneous reaction will proceed where the products have lessenergy than the reactants (substrate) and ΔG is the free energy changebetween the product and the reactant.

• Note that the enzyme does not alter the free energy change in a reaction which

is why it does not alter the equilibrium associated with a reaction - only the rate.

http://www.google.ca/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=YXJhZM15gWZNCM&tbnid=4z9sTnds0mMrfM:&ved=0CAUQjRw&url=http://www.biology.arizona.edu/biochemistry/problem_sets/energy_enzymes_catalysis/01t.html&ei=YVBHUuy0DYW84APbjoCwAg&bvm=bv.53217764,d.dmg&psig=AFQjCNE5taUiaMvrrZOZOHmTEh7mZ280uQ&ust=1380491670042753


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Enzymes lower Activation Energy by stabilizing a Transition Stateintermediate

• the free energy difference between reactants and products determines the

equilibrium for a reaction• but enzymes determine how quickly the equilibrium is reached by helping toform a transition state chemical form that is no longer reactant (substrate) butnot yet product in structural terms

S Xⱡ P where ⱡ denotes transition state

The transition state is a high energy and unstableintermediate that must form during the conversionof substrate to product. It has a very short half-life

(about 1 x 10 -13 s).Its high energy is the source of activation energy(ΔGⱡ).

Enzymes therefore lower activation energy by providing

a chemical scaffold that facilitates the formation ofthe transition state molecule


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Enzymes therefore lower activation energy by providinga chemical scaffold that facilitates the formation ofthe transition state molecule

• when two chemicals react, the ability to form a transition state intermediateand eventual product relies on two main conditions:

• the kinetic energy of the colliding substrate molecules• the orientation of the colliding substrate molecules

• adding an external source of heat energy is one way to provide the requiredkinetic energy to substrate molecules such that they are able to formthe transition state intermediate

•

an enzyme reduces activation energy by binding two reactants, bringingthem physically together in a orientation that permits reaction.

• This occurs in the ACTIVE SITE of the enzyme.


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Substrates bind in the active site of an enzyme

• a relatively small portion of an enzyme is involved in catalysis

• the active site accommodates only specific substrates on the basis of size, shape,and chemical characteristics of the substrate

• the induced fit model describes that both the structure of the enzyme and thesubstrate is changed upon binding

http://www.google.ca/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=Wy3KsSs-EA6rrM&tbnid=sHo6BdbVEoJPKM:&ved=0CAUQjRw&url=http://iws.collin.edu/biopage/faculty/mcculloch/1406/outlines/chapter%206/chap06.html&ei=ZlJHUvubMdGo4AP734GIAg&bvm=bv.53217764,d.dmg&psig=AFQjCNH9gNxhK9x-9KzaBsX7_c-BLR_IYQ&ust=1380492221615696


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Amino acid side-chains interact with substrate molecules through weak forcessuch as ionic, hydrogen-bonding, and van der Waals interactions.

it is these inter-molecular interactions that determine a) binding specificity,b) the orientation of substrate molecules in the active site, c) stabilization of

the transition state structure, d) chemical reactivity or catalysis

http://www.google.ca/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=Wy3KsSs-EA6rrM&tbnid=sHo6BdbVEoJPKM:&ved=0CAUQjRw&url=http://www.daviddarling.info/encyclopedia/A/active_site.html&ei=O1NHUqOTFcPj4APHpICQAQ&psig=AFQjCNH9gNxhK9x-9KzaBsX7_c-BLR_IYQ&ust=1380492221615696http://www.google.ca/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=Wy3KsSs-EA6rrM&tbnid=sHo6BdbVEoJPKM:&ved=&url=http://www.bath.ac.uk/cer/research/metabolism.html&ei=7lJHUpOtO5Wl4AOmrYGoAg&psig=AFQjCNH9gNxhK9x-9KzaBsX7_c-BLR_IYQ&ust=1380492221615696


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Catalytic amino acids reside in the active site

• the active site binding pocket or cleft is where several amino acid side chains thatplay instrumental roles in chemical catalysis coincide

• these are called active site residues or catalytic residues

Naturally, the folding of aprotein into its tertiary stateis not a random process.


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Protein Evolution

• as species diverge from one another, mutations will inevitably and independentlyoccur in the genes that encode proteins.

• some mutations may be lethal and the host organism dies• other mutations are permitted, leading to divergence amongst the amino acid

sequences of like-proteins from different species.

Here are four like-proteins from different bacterial species.These four proteins carry out the same function in each bacterium.

• Note that not all amino acids have similar importance in the protein. Some areimportant for structure (pink) and some are active site catalytic residues (blue) andthese are largely conserved.•

Some amino acids at specific positions are not conserved at all, indicating theyare not overtly important to the protein or enzyme

1 N - MYFGHATFRVVARFPLKMNKYTWYHCNPCKACIYTGDEGGAKIADENS - C

2 N - MDKWAKWFMVAARGLCADDKLAQRHVQLCQACKMAADELNWEIALPAV - C

3 N - MSVKLGCWQVLARDEDSTGKAFERHVEICQACKLMADEAGSEIAYCVV - C

4 N - MDEWAVNFMVVARHLAADQRLANRHVADCQACKAWADDKQMEIVFQAV - C


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Some Enzymes possess co-factors required for either structureor catalysis

• some enzymes function on their own while some have co-factors intimately

associated with their structure

• co-factors may be metal ions or larger molecules called co-enzymes

• both of these often participate in the catalysis process

• co-enzymes are often vitamin derivatives

A Holoenzyme forms

when the Apoenzyme formof the protein binds itsco-factor

http://www.google.ca/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=Gs1VKfJaDJiwJM&tbnid=31pYAK0y2VjyMM:&ved=0CAUQjRw&url=http://lecturer.ukdw.ac.id/dhira/Metabolism/Enzymes.html&ei=HElHUpnHKuam2gW7yoGICQ&bvm=bv.53217764,d.b2I&psig=AFQjCNHLK6UM13IDoS0g5GmoOIg2uqm_7w&ust=1380489528298829


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Some selected co-factors (coenzymes and metal ions)

• for enzymes that require co-factors, the protein lacking the co-factor is inactive• this is the central reason why vitamins and certain metal ions are essential for life

Thiamine, Vit B1Riboflavin, Vit B2Niacin

Vitamin B6Pantothenic acid

Vit B12Folic acid

the 5 essential ionsCa2+, K+, Na+, Mg 2+, Cl-


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The catalytically important amino acids and the co-factors define the active siteof the enzyme and interact with substrate, the transition state intermediateand products formed

Active site amino acid side chains of the enzyme Cytochrome P450and its co-factor Heme interacting with its substrate camphor

http://www.google.ca/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRw&url=http://omlc.org/spectra/hemoglobin/hemestruct/&ei=e_8-VKT1BZGoyATJ34HwDg&bvm=bv.77648437,d.aWw&psig=AFQjCNEXsEQsmqEK9v9ftskJ0x7D-2WDjg&ust=1413501149611094


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Active site amino acid side chains specifically interact with substrate moleculesas shown in the active site of Ribonuclease (an enzyme that hydrolyzes RNAmolecules to free ribonucleotides).

We will talk about the

mechanism by whichenzymes catalyzechemical reaction later


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Next few lectures we will cover:

Enzyme kinetics (the study of the rate of enzyme catalysis)

Types of catalysis

The Chemical Mechanisms of enzymatic catalysis

lecture 11 - introduction to enzymes

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