ENZYMES

ENZYME INHIBITION Inhibitor – substance that binds to an

enzyme and interferes with its activity

Can prevent formation of ES complex or prevent ES breakdown to E + P.

There are 3 categories of enzyme inhibition –

1. Reversible inhibition

2. Irreversible inhibition

3.Allosteric inhibition

REVERSIBLE INHIBITION

Reversible Inhibitors bind through non-covalent interactions (disassociates from enzyme)

E + S <-> ES -> E + PE + I <-> EI

Competitive Uncompetitive Non-competitive

COMPETETIVE INHIBITION Occurs when the inhibitor binds reversibly

to the same site that the S would normally occupy and competes with the S for that site.

Competitive inhibitors are of similar chemical structure to the substrate.

Has effect reversed by increasing substrate concentration

COMPETITIVE INHIBITOR (CI)

•CI binds free enzyme

•Competes with substrate for enzyme binding.

•Raises Km without effecting Vmax

•Can relieve inhibition with more S

COMPETITIVE INHIBITORS CAN BE OVERCOME BY INCREASING [S]

Substrates bind & reaction proceeds

COMPETITIVE INHIBITORS LOOK LIKE SUBSTRATE

NH2C

O

HO NH2S

O

H2N

O

PABA Sulfanilamide

PABA precursor to folic acid in bacteria

Examples: Mehotrexate [anticancer drug] inhibit folate reductase

METHANOL POISONING

Methanol is very toxic and ingestion may lead to optic neuritis, blindness and even death.

Methanol alcohol dehydrogenase Formaldehyde [cause toxicity]

Antidote for methanol poisoning is ethanol [ is a inhibitor of alcohol dehydrogenase]

Ethanol reduces the utilization of methanol and toxicity is averted

Ethylene glycol [antifreeze for automobile] upon ingestion caused CNS depression and Renal damage. Ethanol is used for treatment

UNCOMPETITIVE INHIBITION

An inhibitor which binds only to ES complexes

UI does bind at the active site, but only AFTER the S has bound to the active site so it does not compete with the S.

UNCOMPETITIVE INHIBITOR (UI)

•UI binds ES complex

•Prevents ES from proceeding to E + P or back to E + S.

•Lowers Km & Vmax, but ratio of Km/Vmax remains the same

•Occurs with multisubstrate enzymes

NON-COMPETITIVE INHIBITION

A noncompetitive inhibitor Does not have a structure like substrate Binds to the enzyme but not active site Changes the shape of enzyme and active

site Substrate cannot fit altered active site No reaction occurs Effect is not reversed by adding substrate

NON-COMPETITIVE INHIBITOR (NI)

•NI can bind free E or ES complex

•Lowers Vmax, but Km remains the same

•NI’s don’t bind to S binding site therefore don’t effect Km

•Alters conformation of enzyme to effect catalysis but not substrate binding

NON-COMPETITIVE INHIBITION

Eg.1) Lead poisoning. Pb forms covalent

bonds with SH gps in proteins such as Hemoglobin and causes anemia

2). Poisons like cyanide inhibits cytochrome oxidase in the respiratory chain and causes death

ENZYME INHIBITION IN THE LAB

1/V

1/[S]

no inhibitor

+ inhibitor 1/V

1/[S]

no inhibitor

+ inhibitor

Competitive Non-competitive

Vmax doesn’t change. Km doesn’t change.

1/Vmax

-1/Km

TYPES OF REVERSIBLE ENZYME INHIBITORS

type binding target Km Vmax

Competitive E only =

Noncompetitive E or ES =

Uncompetitive ES only

Summary of inhibition

IRREVERSIBLE INHIBITORS

Irreversible inhibitor binds to enzyme through covalent bonds (binds irreversibly)

IRREVERSIBLE INHIBITORS

H3C O P

O

S C

C

H

O

O CH2CH3

C O CH2CH3

O

S

CH3

CH2

H C

CH3

O

CH3

P

F

O

O C

CH3

H

CH3

Diisopropyl fluorophosphate(nerve gas)

H3C O P

O

S

S

CH3

NO2

parathion

malathion

•Organophosphates•Inhibit serine hydrolases•Acetylcholinesterase inhibitors

IRREVERSIBLE INHIBITION

SUICIDE INHIBITION – Specialized form of irreversible inhibition Original inhibitor (structure analogue) is

converted to more potent form by the same enzyme that ought to be inhibited.

Eg. Allopurinol, an inhibitor of xanthine oxidase, get converted to alloxanthine, a more effective form.

ENZYME SPECIFICITY Enzymes have varying degrees of specificity

for substrates Enzymes may recognize and catalyze:

- a single substrate- a group of similar substrates- a particular type of bond

There are 3 types of enzyme specificity- 1. Stereospecificity 2. Reaction specificity 3. Substrate specificity

1. Stereospecificity

The enzyme can act on only one form of isomers of the substrates.

H

C

H3C COOHOH

H

C

H3C OHCOOH

AB C A

B C

LACTATE DEHYDROGENASE CAN RECOGNIZE ONLY THE L-FORM BUT NOT THE D-FORM LACTATE.

2.REACTION SPECIFICITY Same substrate can undergo different type of

reaction, each catalysed by different enzyme. Amino acid can undergo deamination ,

transamination, etc.

3. SUBSTRATE SPECIFICITY

A. Absolute substrate specificity B. Relative substrate specificity C. Broad specificity

ABSOLUTE SPECIFICITY

Enzymes can recognize only one type of substrate and implement their catalytic functions.

O C

NH2

NH2

+ H2O 2NH3 + CO2

urea

urease

O C

NH

NH2

+ H2O

methyl urea

CH3

Enzymes catalyze one class of substrates or one kind of chemical bond in the same type.

RELATIVE SPECIFICITY

protein kinase Aprotein kinase Cprotein kinase G

To phopharylate the -OH group of serine and threonine in the substrate proteins, leading to the activation of proteins.

OH

OH

HH

OHH

OH

CH2OH

H

CH2OH

HCH2OH

OH H

H OH

O

O

1

1

OH

OH

HH

OHH

OH

CH2

H

CH2OH

HCH2OH

OH H

H OH

O

O

1

1

O

OOH

H

HH

OHH

OH

CH2OH

H 1

sucrose

raffinose

sucrase

BROAD SPECIFICITY

Enzymes acts on closely related substrates E.g. hexokinase acts on glucose,

fructose,mannose

LOCK-AND-KEY MODEL In the lock-and-key model of enzyme action:

- the active site has a rigid shape- only substrates with the matching shape can fit- the substrate is a key that fits the lock of the active site

This is an older model, however, and does not work for all enzymes

INDUCED FIT MODEL In the induced-fit model of enzyme action:

- the active site is flexible, not rigid- the shapes of the enzyme, active site, and substrate adjust to maximumize the fit, which improves catalysis- there is a greater range of substrate specificity

This model is more consistent with a wider range of enzymes

REGULATION OF ENZYME

Many biological processes take place at a specific time; at a specific location and at a specific speed.

The catalytic capacity is the product of the enzyme concentration and their intrinsic catalytic efficiency.

The key step of this process is to regulate either the enzymatic activity or the enzyme quantity.

Maintenance of an ordered state in a timely fashion and without wasting resources

Conservation of energy to consume just enough nutrients

Rapid adjustment in response to environmental changes

REASONS FOR REGULATION

Controlling an enzyme that catalyzes the rate-limiting reaction will regulate the entire metabolic pathway, making the biosystem control more efficient.

Rate limiting reaction is the reaction whose rate set by an enzyme will dictate the whole pathway, namely, the slowest one or the “bottleneck” step.

Zymogen activation

Allosteric regulation

Covalent modification

REGULATION OF ENZYME ACTIVITY

Certain proteins are synthesized and secreted as an inactive precursor of an enzyme, called zymogen.

Selective proteolysis of these precursors leads to conformational changes, and activates these enzymes.

It is the conformational changes that either form an active site of the enzyme or expose the active site to the substrates.

A ZYMOGEN ACTIVATION

Hormones: proinsulin

Digestive proteins: trypsinogen, …

Funtional proteins: factors of blood clotting and clot dissolution

Connective tissue proteins: procollagen

WIDE VARIETIES

A cascade reaction in general

To protect the zymogens from being digested

To exert function in appropriate time and location

Store and transport enzymes

FEATURES OF ZYMOGEN ACTIVATION

Allosteric enzymes are those whose activity can be adjusted by reversible, non-covalent binding of a specific modulator to the regulatory sites, specific sites on the surface of enzymes.

Allosteric enzymes are normally composed of multiple subunits which can be either identical or different.

B ALLOSTERIC REGULATION

The multiple subunits are

catalytic subunits

regulatory subunits

Kinetic plot of v versus [S] is sigmoidal shape.

Demonstrating either positive or negative cooperative effect.

There are two conformational forms, T and R, which are in equilibrium.

Modulators and substrates can bind to the R form only; the inhibitors can bind to the T form.

Properties of allosteric enzymes

[S]

Allosteric enzyme

Allosteric represion

Allosteric activation

ALLOSTERIC CURVE

ACTIVATION OF PROTEIN KINASE

C: catalytic portionsR: regulatory portions

4 cAMP

protein kinase(inactive)

protein kinase(active)

+ +C

C

R

R

C

C

R

R

cAMP

cAMP

cAMP

cAMP

A variety of chemical groups on enzymes could be modified in a reversible and covalent manner.

Such modification can lead to the changes of the enzymatic activity.

C COVALENT MODIFICATION

phosphorylation - dephosphorylation adenylation - deadenylationmethylation - demethylation

uridylation - deuridylationribosylation - deribosylationacetylation - deacetylation

COMMON MODIFICATIONS

Phosphorylation

E-OH E-O-PO3H2

ATP ADP

proteinkinase

phosphorylation

dephosphorylation

H2OPi

Mg2+

phosphatase

Two active forms (high and low)

Covalent modification

Energy needed

Amplification cascade

Some enzymes can be controlled by allosteric and covalent modification.

FEATURES OF COVALENT MODIFICATION

Constitutive enzymes (house-keeping): enzymes whose concentration essentially remains constant over time

Adaptive enzymes: enzymes whose quantity fluctuate as body needs and well-regulated.

Regulation of enzyme quantity is accomplished through the control of the genes expression.

REGULATION OF ENZYME QUANTITY

Inducer: substrates or structurally related compounds that can initiate the enzyme synthesis

Repressor: compounds that can curtail the synthesis of enzymes in an anabolic pathway in response to the excess of an metabolite

Both are cis elements, trans-acting regulatory proteins, and specific DNA sequences located upstream of genes

CONTROLLING THE SYNTHESIS

Enzymes are immortal, and have a wide range of lifetime. LDH4 5-6 days, amylase 3-5 hours.

They degrade once not needed through proteolytic degradation.

The degradation speed can be influenced by the presence of ligands such as substrates, coenzymes, and metal ions, nutrients and hormones.

CONTROLLING THE DEGRADATION

Lysosomic pathway: Under the acidic condition in lysosomes No ATP required Indiscriminative digestion Digesting the invading or long lifetime proteins

Non-lysosomic pathway: Digest the proteins of short lifetime Labeling by ubiquitin followed by hydrolysis ATP needed

DEGRADATION PATHWAY

ENZYMES/PATHWAYS IN CELLULAR ORGANELLES organelle Enzyme/metabolic pathway

Cytoplasm Aminotransferases, peptidases, glycolysis, hexose monophosphate shunt, fatty acids synthesis, purine and pyrimidine catabolism

Mitochondria Fatty acid oxidation, amino acid oxidation, Krebs cycle, urea synthesis, electron transport chain and oxidative phosphorylation

Nucleus Biosynthesis of DNA and RNA

Endoplasmic reticulum

Protein biosynthesis, triacylglycerol and phospholipids synthesis, steroid synthesis and reduction, cytochrome P450, esterase

Lysosomes Lysozyme, phosphatases, phospholipases, proteases, lipases, nucleases

Golgi apparatus Glucose 6-phosphatase, 5’-nucleotidase, glucosyl- and galactosyl-transferase

Peroxisomes Calatase, urate oxidase, D-amino acid oxidase, long chain fatty acid oxidase

UNIT OF ENZYME ACTIVITY

KATAL- mol substrate/sec = katal

INTERNATIONAL UNIT(IU) or STANDARD UNIT(SI unit)

mol substrate transformed/min = unit

1IU = 60 katal

ISOENZYMES

Isoenzymes are different forms of an enzyme that catalyze the same reaction in different tissues in the body- they have slight variations in the amino acid sequences of the subunits of their quaternary structure

For example, lactate dehydrogenase (LDH), which converts lactate to pyruvate, consists of five isoenzymes

ISOENZYMES

ISOENZYMES

EXAMPLES – Isoenzymes of creatine phosphokinase(CPK) or

creatine kinase (CK)it catalyse phosphocreatine to creatineCPK exists in 3 isoenzymes and has 2 subunits M (muscle) , B (brain) or both

CPK1 - BB - brainCPK2 - MB - heartCPK3 - MM - skeletal muscle

APPLICATION OF ENZYMES

A. Enzymes as therapeutic agents – 1. Streptokinase in case of blood clots.

Plasminogen to plasmin in presence of streptokinase then fibrin (clot) to soluble product.

2. Asparaginase in case of leukemias.

B. Enzyme as analytical reagentsuseful in lab.e.g estimation of a plasma glucose by glucose oxidase and peroxidase method

DIAGNOSTIC ENZYMES The levels of diagnostic enzymes in the blood can be used

to determine the amount of damage in specific tissues