ENZYMES

Rachel JacobS2 M.Sc ZoologyZOO-15-05-10

Topics to be covered:

Enzyme Activity

Enzyme Receptors

Regulation of Enzyme Activity

Enzymes are complex proteins

of high molecular weight which catalyze specific

biochemical reactions in organisms .

A peep into the past

• At the end of the nineteenth century, scientist debated on whether the process of ethanol formation required the presence of intact yeast cells.

• Justus von Liebig (an organic chemist) argued that the reaction of fermentation that produced alcohol, were no different in vivo and in vitro.

• Louis Pasteur(the renowned biologist) argued that the process of fermentation can occur only within the confines of an intact living cell.

• Hans Buchner and Eduard Buchner prepared yeast extract which they tried to preserve by adding sugar, found that the extract produced gas from sugar and it bubbled continuously for days.

• They discovered that fermentation produced ethanol and bubbles of carbon dioxide.

• Buchner had shown that fermentation did not require the presence of intact cells.

• Later it was found that fermentation differed from chemical reactions carried out in vitro, in the presence of biological catalysts called Enzymes.

The first evidence that enzymes are proteins was obtained in 1926 by James Sumner of Cornell University when he crystallized the enzyme Urease from Jack Beans.

Later RNA

catalysts were also discovered. They are called ribozymes. But the term enzyme is used to refer to those catalysts which are proteins.

Hammerhead Ribozyme

Properties of Enzymes

They are globular proteins required in small amounts.

They are not altered irreversibly during the reaction.

They have no effect on thermo dynamics of reactions

They are very efficient

They are highly specific and regulated and reversible.

Enzymes increase velocity of reactions 108 to 1013 fold at milder temperature and pH.

TERMINOLOGYComplex protein enzymes have a protein part containing amino acids called apoenzyme .

The non-protein part is called cofactor associated with the protein unit.

These two parts bind together to form a holoenzyme.

Inorganic cofactors are enzyme activators (e.g., Mg2+, Ca2+, Co2+)

Organic enzyme cofactors are called coenzymes.

(e.g., NAD or Coenzyme 1).

Prosthetic groups are cofactors tightly bound to an enzyme on a permanent basis. (Eg., Haem)

Ribozymes are RNA molecules with catalytic properties. Eg., hammerhead ribozyme, the VS ribozyme, Leadzyme and the hairpin ribozyme.

Abzymes are antibodies which express catalytic activity.

A single molecule of an antibody-enzyme, or abzyme, is capable of catalyzing the destruction of thousands of target molecules.

On the basis of the chemical

reactions catalyzed by the

enzymes, six different classes

have been recognized.

Enzyme Activity

Substrate Binding

Initial contact between the active site of an enzyme and a potential substrate molecule depends on their collision.

Substrate binding usually involves hydrogen bonds or ionic bonds Substrate binding is readily reversible.

Diagrammatic representation of the active site of the enzyme ribulose bisphosphate carboxylase oxygenase showing the various sites of

interaction between the bound substrates (RUBP and CO2) and certain amino acid side chains of the

enzyme.

The lock-and-key model, first suggested in 1894 by the German biochemist Emil Fischer, explained enzyme specificity but not the catalytic event.

Tight spatial relationship between a glutamic acid (yellow) and a histidine (green) of the enzyme triosephosphate

isomerase respectively and the substrate (red).

The induced-fit model was first proposed in 1958 by Daniel Koshland.

Substrate binding at the active site distorts enzyme and the substrate, stabilizing the substrates in their transition state.

Critical amino acid side chains are brought into the active site even if they are not nearby in the absence of substrate.

Hexokinase (a) without (b) with glucose substrate

SUBSTRATE ACTIVATION

Active site activates substrates.Binding induces a conformational change.

Substrate orientation

Charge transfer

Distorts the substrate

Substrate orientationEnzymes lower the etropy of the system Substrates brought very close together in precisely the correct orientation to undergo reaction.

Changing Substrate Reactivity

Enzymes side chains may be polar or non-polar which influence distribution of electrons in substrate.

Inducing Strain on the Substrate

• As conformational changes occur in the substrate, mechanical work is performed, exerting a physical force on certain bonds within a substrate molecule.

CATALYTIC EVENT The sequence of events at the active site is illustrated using

the enzyme sucrase as an example.

Sucrase (also known as invertase or b-fructofuranosidase) hydrolyzes the disaccharide sucrose into glucose and fructose.

The initial random collision of a substrate molecule—sucrose, in this case—with the active site results in its binding to amino acid residues that are strategically positioned there.

Substrate binding induces a change in the enzyme conformation that tightens the fit between the substrate molecule and the active site and lowers the free energy of the transition state.

This facilitates the conversion of substrate into products—glucose and fructose, in this case.

The products are then released from the active site, enabling the enzyme molecule to return to its original conformation, with the active site now available for another molecule of substrate.

This entire sequence of events takes place in a sufficiently short time to allow hundreds or even thousands of such reactions to occur per second at the active site of a single enzyme molecule.

ENZYME ACTIVITYEnzyme Activity is Regulated

by the Following Factors:

Environmental Conditions

Cofactors and Coenzymes

Enzyme inhibitors

ENVIRONMENTAL CONDITIONS

Temperature

pH

Substrate concentration

Temperature• Q10 (the temperature coefficient) = the increase

in reaction rate with a 10°C rise in temperature.

• For chemical reactions the Q10 = 2 to 3(the rate of the reaction doubles or triples with every 10°C rise in temperature).

• Enzyme-controlled reactions follow this rule as they are chemical reactions.

• BUT at high temperatures proteins denature.

• The optimum temperature for an enzyme controlled reaction will be a balance between the Q10 and denaturation.

Temperature / °C

Enzyme activity

0 10 20 30 40 50

Q10 Denaturation

• For most enzymes the optimum temperature is about 30°C.

• Many are a lot lower, cold water fish will die at 30°C because their enzymes denature.

• A few bacteria have enzymes that can withstand very high temperatures up to 100°C.

• Most enzymes however are fully denatured at 70°C.

p H• Extreme pH levels will produce denaturation.

• The structure of the enzyme is changed.

• The active site is distorted and the substrate molecules will no longer fit in it.

• At pH values slightly different from the enzyme’s optimum value, small changes in the charges of the enzyme and it’s substrate molecules will occur.

• This change in ionisation will affect the binding of the substrate with the active site.

p H

Enzyme activity Trypsin

Pepsin

pH1 3 5 7 9 11

Substrate Concentration For non-enzymatic reactions

Reaction velocity

Substrate concentration

The increase in velocity is proportional to the substrate concentration.

Enzyme catalyzed reaction

Reaction velocity

Substrate concentration

Vmax

Faster reaction but it reaches a saturation point when all the enzyme molecules are occupied.If you alter the concentration of the enzyme then Vmax will change too.

ENZYME INHIBITORS

Inhibition of enzyme activity is important for several reasons.

o It plays a vital role as a control mechanism in cells.

o Enzyme inhibition is also important in the action of drugs and poisons.

o Inhibitors are useful as tools in studies of reaction mechanisms and for treatment of diseases.

Especially important inhibitors are substrate analogues and transition state analogues.

Mechanism of action of substrate analogues

Substrate analogs are used against infectious disease.

For example, sulfa drugs resemble the folic acid precursor, PABA.

• Azidothymidine (AZT) resembles the deoxythymidine molecule used by HIV to synthesize DNA using viral reverse transcriptase.

• After binding to the active site, AZT forms a “dead-end” molecule of DNA that cannot be elongated.

Inhibitors may be either reversible or

irreversible.

IRREVERSIBLE INHIBITORS

• An irreversible inhibitor binds covalently, causing permanent loss of catalytic activity and are toxic.

• Ions of heavy metals are often irreversible inhibitors, as are some pesticides and nerve gas poisons.

• These can bind irreversibly to enzymes such as acetylcholinesterase.

• Inhibition leads to rapid paralysis of vital functions and therefore to death. E.g., diisopropylflourophosphate.

• Diisopropyl fluorophosphate rends the enzyme molecule permanently inactive.

• Some irreversible inhibitors of enzymes can be used as therapeutic agents.

• For example, aspirin binds irreversibly to the enzyme cyclooxygenase-1 (COX-1), which produces prostaglandins.

• Thus, aspirin is used in low doses as a cardiovascular protectant.

The antibiotic penicillin is an irreversible

inhibitor of the enzyme needed

for bacterial cell wall

synthesis.

Reversible Inhibitor A reversible inhibitor binds to an enzyme in a

noncovalent, dissociable manner, such that the free and bound forms of the inhibitor exist in equilibrium with each other.

The two most common forms of reversible inhibitors are called competitive inhibitors and noncompetitive inhibitors.

A competitive inhibitor binds to the active site of the enzyme and therefore competes directly with substrate molecules for the same site on the enzyme.

Competitive Inhibition• Competitive inhibitors are reversible inhibitors that

compete with a substrate for access to the active site of an enzyme.

• Competitive inhibitors must resemble the substrate to compete for the same binding site, but differ in a way that prevents them from being transformed into product.

• Angiotensin converting enzyme (ACE) is a proteolytic enzyme that acts on a 10-residue peptide (angiotensin I) to produce an 8-residue peptide (angiotensin II).

• Elevated levels of angiotensin II are a major risk factor in the development of high blood pressure (hypertension).

• In the 1960s John Vane and his colleagues at the Eli Lilly Company began a search for compounds that could inhibit ACE.

• Brazilian pit viper contains inhibitors of proteolytic enzymes, and it was found that one of the components of this venom, a peptide called teprotide was a potent competitive inhibitor of ACE.

• It was not a very useful drug because it had a peptide structure and thus was rapidly degraded if taken orally.

• Subsequent efforts to develop nonpeptide inhibitors of the enzyme led researchers to synthesize a compound called captopril, which became the first useful antihypertensive drug that acted by binding to ACE.

Given a sufficient substrate concentration, it remains theoretically possible to achieve the enzyme’s maximal velocity even in the presence of the competitive inhibitor.• Hence maximal reaction velocity can be

achieved (Vmax remains unchanged)• But the enyme’s affinity to the substrate

is reduced ( Km increases)

Non- competitive inhibition• In noncompetitive inhibition, the substrate

and inhibitor do not compete for the same binding site; generally, the inhibitor acts at a site other than the enzyme’s active site.

• The level of inhibition depends only on the concentration of the inhibitor, and increasing the concentration of the substrate cannot overcome it.

Since, in the presence of a noncompetitive inhibitor, a certain fraction of the enzyme molecules are necessarily inactive at a given instant, the maximal velocity of the population of enzyme molecules cannot be reached.

• Tipranavir is a potent noncompetitive inhibitor of the protease produced by HIV when it infects a white blood cell.

• Unlike other inhibitors of this enzyme, such as ritonavir, tipranavir does not resemble the peptide substrate of the enzyme, nor does it compete with the substrate.

Considerable progress has been made in the field of computer-aided drug design.

Scientists can now design a number of hypothetical inhibitors and test their binding using complex computer models.

ENZYME KINETICS

• Chemical kinetics studies the rate of chemical reactions and the factors influencing them.

• Similarly, enzyme kinetics studies the rate of enzyme catalyzed reactions and the factors influencing them.

Canadian physician Maud Leonara Menten and German biochemist

Leonor Michaelis and (1913) proposed a general theory to explain

enzyme action.

Maud Menten and Leonor Michaelis

They tried to explain the relationship between reaction rate and substrate concentration.

Assumptions They assumed a steady state to pre-exist.

The reaction is in a “steady state” when the concentration of the enzyme substrate

complex remains steady.

The MM equation concerns the initial velocity of the reaction (v0) when the rate

of the backward reaction is negligible.

Wherein v0 = initial reaction velocity

[S] = initial substrate concentrationVmax = Maximum velocity

Km = substrate conc. at ½ Vmax

Plots of the initial velocity at different concentrations of the enzyme as a function of substrate concentration.

The maximal velocity Vmax is quadrupled once the enzyme concentration in quadrupled. The Km remains unaltered.

Plots of the initial velocity versus substrate concentration for a Substrate S for which the enzyme has higher affinity and a substrate S’ for which the enzyme has lower affinity.

Vmax remains same for the same enzyme, but higher the Km, lower the affinity

• Km is a measure of affinity of the enzyme for its substrate. Greater the Km, lesser is the affinity.

• Rule of thumb :Intracellular concentration of a substrate is approximately the same as, or approximately greater than the Km value of the enzyme to which it binds.

Can you crack this ?

From CSIR Dec 2014

And the right answer is..Option A

Did you get it right?If so, Congrats!

If not, don’t worry. A little practice will set you in the right

track .

Case 1

At low substrate concentration:

v0= Vmax [S]

Km

Case 2• At intermediate

substrate concentrations,

[S]= Km

V0=Vmax/2

Case 3

At high substrate concentrations:

V0=Vmax

Lineweaver-Burk Plot

• MM equation yields a hyperbola.

• Accurate prediction of Km and Vmax values from this hyperbola requires a large number of data points.

• The Lineweaver Burk plot or the double reciprocal plot helps overcome this.

• It is simply the reciprocal of MM equation.

• It enables accurate prediction of Km and Vmax from a few data points and extrapolation becomes easier.

Effect of inhibitors on kinetics

Enzyme Receptors

Enzyme linked receptors form a major type of cell-surface receptors.

Enzyme-linked receptor proteins either possess an in-built enzyme or associate with separate enzymes in the cytoplasm.

These enzymes are activated upon ligand binding.

They relay the extracellular signal to the nucleus by a sequence of interactions.

This turns on specific transcription factors, altering gene expression in the cell.

BASIC STRUCTURE

All enzyme-linked receptors share a few common features;

1. Ligand-binding domain• Extracellular to allow easy access for ligands.

• Strong affinity for specific ligands - allows different ligands that bind to the same receptor to evoke particular

cellular responses. 

2. Transmembrane domain• Contains a series of hydrophobic amino acids.• Tethers the receptor to the cell membrane.

 3. Cytosolic "active" enzyme domain• Either intrinsic to the receptor or tightly bound via the

cytosolic domain.• The majority are kinases; they phosphorylate specific

threonine, serine, and tyrosine amino acid residues (THR,S,TY = THIRSTY).

Enzyme-linked receptor classes

There are three main types of enzyme-linked receptors:

• 1. Receptor serine-threonine kinases e.g. transforming growth factor-beta (TGFB) receptors. 

• 2. Receptor tyrosine kinases (RTKs) e.g. growth factor receptors.

• 3. Tyrosine-kinase-associated receptors e.g. cytokine receptors. 

Receptor serine/threonine kinases

• There are two units of serine/threonine kinase receptors, both of which contain an intracellular kinase domain. They are each dimeric proteins, so an active receptor complex is made up of four units.

• 1. Type I receptors– Inactive unless in complex with type II receptors.– Do not interact with ligand dimers.– Contain conserved sequences of serine and threonine residues near to

their kinase domains. 

• 2. Type II receptors– Constitutively active kinase domains (even in the absence of the

bound ligand).– Able to phosphorylate and activate the type I receptor.

• Type I receptors are kept inactive by a portion of its cytosolic domain that blocks its kinase activity.

• Type II receptors binds to, and phosphorylate, Type I receptors. This removes the inhibition of Type I kinase activity. 

• Type I receptors then phosphorylate Smad transcription factors which dimerize & enter the nucleus to repress or activate target gene expression.

Receptor Tyrosine Kinase RTK ligands, such as fibroblast growth factor (FGF), epidermal

growth factor (EGF), nerve growth factor (NGF) etc. bind as dimers.

1.Ligand binding to RTK monomers results in dimer formation.

2.Receptors possess an intracellular tyrosine kinase domain. Within the dimer the conformation is changed, locking the kinase into an active state.

3.The kinase of one receptor then phosphorylates a tyrosine residue contained in the second receptor.

4.Phosphorylated tyrosines function as binding sites for intracellular signalling proteins.

Tyrosine Kinase Associated Receptors

• Cytokines are the main ligands that signal through tyrosine kinase-associated receptors. 

• Like RTKs and RS/TKs these receptors activate a cascade of phosphorylation BUT they do not possess a tyrosine kinase domain.

• The intracellular side of each receptor is bound to a cytosolic tyrosine kinase protein.

 

Tyrosine Kinase Associated Receptors

• 1. Cytokines bind simultaneously to two receptor monomers.

• 2. This brings the two associated kinases closer together.

• 3. One kinase phosphorylates the other kinase.

• 4. The enhanced kinase phosphorylates more tyrosine residues on the intracellular portion of the receptor.

• 5. Phosphotyrosines serve as "docking sites" for SH2 domain-containing proteins.

Enzyme Regulation

Enzyme regulation is the process, by which cells can turn on, turn off, or modulate the activities of various metabolic pathways. The four kinds of enzyme regulation are

Allosteric regulationReversible covalent modification

Proteolytic activationRegulation by control proteins

Allosteric Regulation

Allosteric regulation is the regulation of an enzyme or other protein by binding an effector molecule at the protein's allosteric site (that is, a site other than the protein's active site).

Reversible Covalent Modification

• The activities of some regulatory enzymes are modulated by reversible covalent modification of the enzyme molecule.

• These include the phosphorylation, adenylation, acetylation, uridylation, ADP-ribosylation, and methylation of enzymes.

Phosphorylation

• Phosphorylation is the most common type of regulatory modification found in eukaryotes.

• Some enzymes are phosphorylated on a single amino acids while others are phosphorylated at multiple sites.

• The attachment of phosphoryl groups is catalyzed by protein kinases;

• Removal of phosphoryl groups is catalyzed by protein phosphatases.

• The phosphoryl groups are attached to serine, threonine , histidine or tyrosine residues.

Adenylation• Adenylylation : the process in which

AMP is covalently attached to a protein, nucleic acid, or small molecule via a phosphodiester linkage.

• In the process of deadenylylation, AMP is removed from the adenylylated molecule.

Uridylation• Uridylylation is the process in which,

a uridylyl group is introduced into a protein, a ribonucleic acid, or a sugar phosphate, generally through the action of a uridylyl transferase enzyme.

ADP-Ribosylation ADP-ribosylation is the addition of one or

more ADP-ribose moieties to a protein. It is observed in only a few proteins; the

ADP-ribose is derived from nicotinamide adenine dinucleotide

(NAD).

Methylation• Methylation is the addtion of a

methyl group to a protein.

• Methyl-accepting chemotaxis protein (MCP) is a transmembrane sensor protein of bacteria.

• With the help of the MCPs, the bacteria can detect concentrations of molecules in the extracellular matrix, as a result of which the bacteria may smooth swim or tumble.

Proteolytic Activation• The activation of an enzyme by peptide

cleavage is known as proteolytic activation.

• In this enzyme regulation process, the enzyme is shifted between the inactive and active state.

• Irreversible conversions can occur on inactive enzymes to become active.

• This inactive precursor is known as a zymogen or a proenzyme, which is cleaved to form the active enzyme.

• The cleavage is independent of ATP.

• Proteolytic activation occurs just once in an enzyme's lifetime.

• So this type of enzyme regulation is also termed as irreversible covalent modification.

Regulation by Control Molecules

• Phosphoprotein phosphatases catalyse the reverse process of protein phosphorylation. Cyclic AMP, an intercellular messenger, can activate protein kinase A (PKA).

• In the absence of cAMP, Protein Kinase A (PKA) exists as an equimolar tetramer of regulatory (R) and catalytic (C) subunits.

• Cyclic AMP activates Protein Kinase A by altering the quaternary structure.

Thank you!!