enzymes 1. history of enzymes -1700s and early 1800s, the digestion of meat by stomach secretions...

ENZYMES

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History of Enzymes

-1700s and early 1800s, the digestion of meat by stomach secretions and the conversion of starch to sugars by plant extracts and saliva were known.--mechanism by which this occurred had not been identified.

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-19th century, when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur came to the conclusion that it was catalyzed by a vital force contained within the yeast cells called "ferments", which were thought to function only within living organisms.

--He wrote that "alcoholic fermentation is an act correlated with the life and organization of the yeast cells, not with the death or putrefaction of the cells.

History of Enzymes

Yeast 3

1878: German physiologist Wilhelm Kühne first used the term enzyme, which literally means “in

yeast” 1897: Eduard Buchner

began to study the ability of yeast extracts that lacked any living yeast cells to ferment sugar. He also found that the sugar was fermented even when there were no living yeast cells in the mixture.

He named the enzyme that brought about the fermentation of sucrose "zymase". In 1907 he received the Nobel Prize in Chemistry “for his biochemical research and his discovery of cell-free fermentation".

History of Enzymes

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Enzymes Enzymes are biomolecules that

catalyze, increase the rates of chemical reactions without being altered during the reaction. Almost all enzymes are proteins; Enzymes are essential to life.

In enzymatic reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, the products.

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Almost all processes in a biological cell need enzymes in order to occur at significant rates.

Since enzymes are extremely selective for their substrates and speed up only a few reactions from among many possibilities, the set of enzymes made in a cell determines which metabolic pathways occur in that cell.

Enzymes

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Enzymes Enzyme activity can be

affected by other molecules. Inhibitors are molecules

that decrease enzyme activity.

Inducers are molecules that increase activity. Many drugs and poisons are enzyme inhibitors.

Activity is also affected by temperature, chemical environment (e.g. pH),

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Enzyme Inhibitor & Inducer

Enzyme Inhibitor Enzyme InducerCimetidine Rifampicin

Ketoconazole Carbamazepine

Fluconazole Phenobarbital

Miconazole Phenytoin

Macrolides(except Azithromycin)

Griseofulvin

Fluoroquinolones(except Levofloxacin)

Smoking

Chronic alcoholism8

ENZYMES are biological catalystENZYME CHARACTERISTICS1. The basic function of an enzyme is

to increase the rate of a reaction 2. Most enzymes act specifically with

only one reactant (called a substrate) to produce products

3. The most remarkable characteristic is that enzymes are regulated from a state of low activity to high activity and vice versa

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Enzymes Lower a Reaction’s Activation Energy

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Enzyme Action

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Three-dimensional structure of an ENZYME

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Enzymes are proteins

They have a globular shape

A complex 3-D structure

Three-dimensional structure of an ENZYME

Human pancreatic amylase

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Enzyme Structure Most enzymes are proteins Enzymes may require a non-peptide

component as a cofactor. The peptide component is called the apoenzyme, the cofactor is called as the coenzyme and the combined functional unit is the holoenzyme

Cofactors that are tightly bound to the polypeptide are called prosthetic groups. Such proteins are called as complex or conjugated proteins. Proteins without prosthetic groups are simple proteins

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The Active Site One part of an

enzyme, the active site, is particularly important

The shape and the chemical environment inside the active site permits a chemical reaction to proceed more easily

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APOENZYME May be inactive in its original synthesized

structure

PROENZYME OR ZYMOGEN The inactive form of the apoenzyme May contain several extra amino

acids in the protein which are removed, and allows the final specific tertiary structure to be formed before it is activated as an apoenzyme

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The Substrate The substrate of an enzyme are the reactants

that are activated by the enzyme; Enzymes are specific to their substrates; The specificity is determined by the active site.

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An additional non-protein molecule that is needed by some enzymes to help the reaction

Nitrogenase enzyme with Fe, Mo and ADP cofactors

COFACTOR

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COFACTOR

A non-protein substance which may be organic and called coenzyme

Common coenzymes are vitamins and metal ions

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COFACTOR Another type of cofactor is an inorganic

metal ion called a metal ion activator Are inorganic and may be bonded through

coordinate covalent bonds Metal ions as Zn+2, Mg+2, Mn+2, Fe+2, Cu+2, K+,

and Na+1 are used in enzymes as cofactors

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Vitamins as Coenzymes

Vitamin Coenzyme Function

Niacin

nicotinamide adenine

dinucleotide (NAD+)

oxidation or hydrogen transfer

Riboflavin flavin adenine

dinucleotide (FAD)

oxidation or hydrogen transfer

Pantothenic acid

coenzyme A (CoA) Acetyl group

carrier

Vitamin B12 coenzyme B-12 Methyl group

transfer

Thiamine (B1) thiaminpyrophosph

ate (TPP) Aldehyde group

transfer

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PROSTHETIC GROUPS

Are tightly incorporated into protein structure by covalent or noncovalent forces

Examples include derivatives of B vitamins such as pyridoxal phosphate, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), thiamin pyrophosphate, biotin and METAL IONS of Co, Cu, Mg, Mn, and Zn.

METALLOENZYMES – enzymes that contain tightly bound metal ions

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PROSTHETIC GROUPS

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NOMENCLATURE

The commonly used names for most enzymes describe the type of reaction catalyzed, followed by the suffix –ase. Dehydrogenases – remove hydrogen

atoms Proteases – hydrolyze proteins Isomerases – catalyze rearrangement

in configuration25

Modifiers may precede the name to indicate; (a) the substrate (xanthine oxidase)(b) the source of the enzyme (pancreatic ribonuclease)(c) its regulation (hormone-sensitive lipase)(d) a feature of its mechanism of action (cysteine protease)

NOMENCLATURE

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Alphanumeric designators may be added to identify multiple forms of an enzyme ( eg., RNA polymerase III; protein kinase C )

Some enzymes retain their original trivial names, which give no hint of the associated enzymatic reaction Examples are pepsin, trypsin, and

chymotrypsin which catalyzes the hydrolysis of proteins

NOMENCLATURE

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Classification of Enzymes

Based on catalyzed reactions, the nomenclature committee of the International Union of Biochemistry and Molecular Biology (IUBMB) recommended the following classification:

1. OXIDOREDUCTASES Catalyze a variety of oxidation-reduction

reactions Common names include dehydrogenase,

oxidase, reductase and catalase

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2. TRANSFERASES Catalyze transfers of

groups (acetyl, methyl, phosphate, etc.).

The first three subclasses play major roles in the regulation of cellular processes.

The polymerase is essential for the synthesis of DNA and RNA.


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Three major regulatory chemical reactions. (a) Acetylation - addition of an acetyl group to lysine's R group by acetyltransferase. (b) Methylation - addition of a methyl group to DNA's

base (e.g. cytosine) by methylase. (c) Phosphorylation - addition of a phosphate group to the R group of tyrosine, serine or threonine (only tyrosine is shown here) by protein kinase.31

3. HYDROLASES Catalyze hydrolysis reactions where a

molecule is split into two or more smaller molecules by the addition of water

PROTEASES split protein molecules HIV protease is essential for HIV replication Caspase plays a major role in apoptosis

NUCLEASES split nucleic acids (DNA and RNA) Based on the substrate type, they are divided into

RNase and DNase. RNase catalyzes the hydrolysis of RNA DNase acts on DNA


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Nucleases cont… They may also be divided into exonuclease and

endonuclease. The exonuclease progressively splits off single

nucleotides from one end of DNA or RNA. The endonuclease splits DNA or RNA at internal sites.

PHOSPHATASE catalyzes dephosphorylation (removal of phosphate groups). Example: calcineurin (also known as protein

phosphatase 3) The immunosuppressive drugs Tacrolimus,

Sirolimus, Everolimus and Cyclosporin A are the calcineurin inhibitors


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4. LYASES

Catalyze the cleavage of C-C, C-O, C-S and C-N bonds by means other than hydrolysis or oxidation.

Common names include decarboxylase and aldolase.

5. ISOMERASES

Catalyze atomic rearrangements within a molecule.

Examples include rotamase, protein disulfide isomerase (PDI), epimerase and racemase


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The role of rotamase and protein disulfide isomerase (PDI). The reactions

catalyzed by the two enzymes can assist a peptide chain to fold into a correct three-dimensional structure

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6. LIGASES Catalyze the reaction which joins two molecules Examples include peptide synthase, aminoacyl-

tRNA synthetase, DNA ligase and RNA ligase

The IUBMB committee also defines subclasses and sub-subclasses

Each enzyme is assigned an EC (Enzyme Commission) number For example, the EC number of catalase is EC1.11.1.6 The first digit indicates that the enzyme belongs to oxidoreductase (class 1)Subsequent digits represent subclasses (1.11. acting on a peroxide as acceptor) and sub-subclasses (1.11.1peroxidases)

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Mechanism of Enzyme Action

The molecule acted upon

a unique geometric shape that is

complementary to the geometric

shape of a substrate molecule

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Lock and Key Theory first postulated in

1894 by Emil Fischer The lock is the

enzyme and the key is the substrate

Only the correctly sized key (substrate) fits into the key hole (active site) of the lock (enzyme)

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The Lock and Key Hypothesis

Enzyme may be used again

Enzyme-substrate complex

E

S

P

E

E

P

Reaction coordinate 40

The Induced Fit Theory

Postulated by Daniel Koshland

It states that, when substrates approach and bind to an enzyme they induce a conformational change

This change is analogous to placing a hand (substrate) into a glove (enzyme)

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The Induced Fit Theory Some proteins can change their shape

(conformation) When a substrate combines with an

enzyme, it induces a change in the enzyme’s conformation

The active site is then moulded into a precise conformation

Making the chemical environment suitable for the reaction

The bonds of the substrate are stretched to make the reaction easier (lowers activation energy)

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This explains the enzymes that can react with a range of substrates of similar types

Hexokinase (a) without (b) with glucose substratehttp://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html

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Assumes that the substrate plays a role in determining the final shape of the enzyme and that the enzyme is partially flexible.

This explains why certain compounds can bind to the enzyme but do not react because the enzyme has been distorted too much

Other molecules may be too small to induce the proper alignment and therefore cannot react

Only the proper substrate is capable of inducing the proper alignment of the active site


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This is a molecular model of the unbound carboxypeptidase A

enzyme

This is a representation of carboxypeptidase A with a

substrate (turquoise) bound in the active site. The active site is

in the induced conformation. 45

MECHANISMS TO FACILITATE CATALYSIS

A. CATALYSIS BY PROXIMITY For molecules to react, they must come within

bond-forming distance of one another The higher the concentration, the more

frequently they will encounter one another and the greater will be their rate of interaction

aka entropy reduction ACID-BASE CATALYSIS

Can be specific or general “Specific” meaning only protons (H3O+ ,

specific acid) or OH- ions (specific base)

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Proximity: Reaction between bound molecules doesn't require an improbable collision of 2 molecules -- they're already in "contact" (increases the local concentration of reactants).

Orientation: Reactants are not only near each other on enzyme, they're oriented in optimal position to react, so the improbability of colliding in correct orientation is taken care of.


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Rate enhancement by entropy reduction.

a) bimolecular reaction (high activation energy, low rate)

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b) unimolecular reaction, rate enhanced by factor of 105 due to increased probability of collision/reaction of the 2 groups.


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c) constraint of structure to orient groups better (elimination of freedom of rotation around bonds between reactive groups), rate enhanced by another factor of 103, for 108 total rate enhancement over bimolecular reaction.


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When substrate binds to enzyme, water is usually excluded from active site (desolvation). causes local dielectric constant to be lower,

which enhances electrostatic interactions in the active site, and also

results in protection of reactive groups from water, so water doesn't react to form unwanted biproducts.

Of course, if water is a substrate, it has to be "allowed in", but maybe only in a certain sub-part of active site.

Involvement of charged enzyme functional groups in stabilizing otherwise unstable intermediates in the chemical mechanism can also correctly be called "electrostatic catalysis".


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CATALYSIS BY STRAIN Strain is created by binding to substrates in a

conformation slightly unfavorable for the bond to undergo cleavage

The strain stretches or distorts the targeted bond, weakening it and making it more vulnerable to cleavage

probably the most important rate enhancing mechanism available to enzymes

Enzyme binds transition state of the reaction more tightly than either the substrate or product --therefore DG‡ is reduced, and rate is enhanced.

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Strain "Strain" is a classic concept in which it was

supposed that binding of the substrate to the enzyme somehow caused the substrate to become distorted toward the transition state. It's unlikely that there is enough energy available in substrate binding to actually distort the substrate toward the transition state.

It's possible that the substrate and enzyme interact unfavorably and this unfavorable interaction is relieved in the transition state.

It's more likely that the enzyme is strained, as for example in induced fit.

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Transition state stabilization is a more modern concept: it is not the substrate that is distorted but rather that the transition state makes better contacts with the enzyme than the substrate does, so the full binding energy is not achieved until the transition state is reached.

Induced fit assumes that the active site of an enzyme is not complementary to that of the transition state in the absence of the substrate. Such enzymes will have a lower value of kcat/Km, because some of the binding energy must be used to support the conformational change in the enzyme. Induced fit increases Km without increasing kcat.


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COVALENT CATALYSIS Involves the formation of a covalent

bond between the enzyme and one or more substrates

Introduces a new reaction pathway with lower activation energy thus faster than the reaction pathway in homogenous solution

Common among enzymes that catalyze group transfer reactions

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ENZYME KINETICSThe field of biochemistry

concerned with the quantitative measurement of the rates of enzyme-catalyzed reactions and the systematic study of factors that affect

these rates5656

ENZYME KINETICSREACTION MODEL

5757

where S is the substrate

E is the enzyme

ES is the enzyme-substrate complex

k1, k-1, and k2 are rate constants

MICHAELIS MENTEN EQUATION

Describes how reaction velocity varies with substrate concentration

vo = Vmax SKm + S

where Vo = initial reaction velocity

Vmax = maximal velocity

Km = Michaelis constant (k-1 + k2)/k1

S= substrate concentration

5858

ASSUMPTIONS1. Relative concentrations

of E and S S >E, so [ES] at any time is small

2. Steady-state assumption– [ES] does not change in time– E + S = ES = E + P, the rate of formation of ES is

equal to that of the breakdown of ES

3. Initial velocity– Used in the analysis of enzyme reactions– Rate of reaction is measured as soon as E and S

are mixed P is very small, the rate of back reaction from P

to S can be ignored5959

CONCLUSIONS1. Characteristics of Kma. Small Km

reflects high affinity of the E for S because a low concentration of S is needed to half-saturate the enzyme – that is, reach a velocity that is ½ Vmax

b. Large Km

Reflects low affinity of E for S because a high concentration of S is needed to half-saturate the enzyme6060

Effect of substrate concentration on reaction

velocities

Small Km for enzyme 1 reflects a high affinity of enzyme for the substrateLarge Km for enzyme 2 reflects low affinity of enzyme for the substrate

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CONCLUSIONS2. Relationship of velocity to enzyme

concentrationThe rate of reaction is directly proportional to the enzyme concentration at all substrate concentrations

3. Order of reactionFirst order - S < Km, the velocity of reaction is roughly proportional to the enzyme concentrationZero order - S > Km, the velocity is constant and equal to Vmax; the rate of reaction is then independent of substrate concentration

6262

At low concentration of substrate( [S]<<Km), The velocity of the reaction is first order – that is, proportional to substrate concentration

At

At high concentration of substrate( [S]>>Km), The velocity of the reaction is zero order – that is, constant and independent OF substrate concentration

6363

Lineweaver-Burk Plot

Also called a double-reciprocal plotIf 1/v0 is plotted VS 1/[S], a straight line is obtainedThe intercept on the x-axis is equal to -1/Km

The intercept on the y-axis is equal to 1/Vmax

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Lineweaver-Burk Plot

Can be used to calculate Km and Vmax as well as to determine the mechanism of enzyme inhibitorsEquation describing the Lineweaver-Burk Plot is:

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INHIBITION OF ENZYME ACTIVITY

INHIBITOR – substance that can diminish the velocity of an enzyme catalyzed reaction

TYPES OF INHIBITION:1. COMPETITIVE INHIBITION2. NONCOMPETITIVE INHIBITION

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COMPETITIVE INHIBITION Inhibitor binds

reversibly to the same site that the substrate would normally occupy, and therefore competes with the substrate for that site

Inhibitors tend to resemble the structures of a substrate, and thus are termed as substrate analogs

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COMPETITIVE INHIBITION Malonate Malonate

(¯O(¯OCOCOCHCH22COO¯) COO¯) competes with Succinate competes with Succinate (¯OOC(¯OOCCHCH22CHCH22COO¯) COO¯) for the active site of for the active site of succinate succinate dehydrogenase (SDH)dehydrogenase (SDH)

SDH catalyze the SDH catalyze the removal of one H atom removal of one H atom from each of the 2 from each of the 2 methylene C’s of methylene C’s of succinatesuccinate

Succinate

Malonate

SDH

SDH

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Succinate

(¯OOC-CH2-CH2-COO¯)

Fumarate

(¯OOC-HC=CH-COO¯)

-2H

Malonate – Enzyme Complex

NO REACTION69

Consequences of competitive inhibition

Vmax is unchanged: At high levels of substrate all of the inhibitor is displaced by substrate.

Km is increased: Higher substrate concentrations are required to reach the maximal velocity.

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NONCOMPETITIVE INHIBITION

Inhibitor and substrate bind at different sites on the enzyme

The inhibitor binds to both E and ES

The noncompetitive inhibitor binds to an allosteric site (different location than the active site) of an enzyme

The binding of an inhibitor to the allosteric site alters the shape of the enzyme, resulting in a distorted active site that does not function properly.

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Effect of Enzyme inhibition on Lineweaver-

Burk plot

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NONCOMPETITIVE INHIBITION

Vmax is decreased: At high levels of substrate the inhibitor is still bound.

Km is not changed: Noncompetitive inhibitors do not interfere the binding of substrate to enzyme

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FACTORS AFFECTING ENZYME REACTIONS

I. SUBSTRATE CONCENTRATION

• The rate of enzyme catalyzed reaction increases with substrate concentration until a maximal velocity (Vmax) is reached

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Effect of Temperature

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The rate of enzyme-catalysed reactions increases as the temperature rises to the optimum temperature

Above a certain temperature, activity begins to decline because the enzyme begins to denature

Enzymes are usually damaged

above about 45°C

Effect of pHEffect of pH

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Each enzyme has an optimal pH

In order to interact, the E and S have specific chemical groups in ionized or unionized state

Amino group in protonated form (-NH3

+) increase catalytic activity

At alkaline pH, amino group is deprotonated decrease in rate of reaction

Extremes of pH can lead to denaturation

REGULATION OF ENZYME ACTIVITY

A. ALLOSTERIC REGULATIONB. REGULATION OF ENZYMES BY COVALENT MODIFICATION

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A. ALLOSTERIC REGULATION

EFFECTORS – molecules that regulate allosteric enzymes that bind noncovalently at a site other than the active site Negative effectors – inhibit enzyme

activity Positive effectors – increases enzyme

activity

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HOMOTROPIC EFFECTORS

Substrate itself serves as an effector Most often a positive effector The presence of a substrate

molecules at one site on the enzyme enhances the catalytic properties of the other substrate-binding sites(their sites exhibit cooperativity)

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HETEROTROPIC EFFECTORS The effector may be different from the

substrate

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Feedback Inhibition

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B. REGULATION OF ENZYMES BY COVALENT MODIFICATION

Most frequently by the addition or removal of phosphate group from specific Ser, Thr, and Tyr residues of the enzyme

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ADPATP

Enzyme-OH Enzyme-OPO3=

HPO4= H2O

Protein phosphatase

Protein kinase

Response of Enzyme to phosphorylation Phosphorylated form may be more or

less active than the unphosphorylated enzyme

Glycogen phosphorylase (degrades glycogen) activity is increased low activity (E), high activity (EP)

Glycogen synthase (synthesize glycogen) activity is decreased low activity (EP), high activity (E)

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INDUCTION and REPRESSION

of enzyme synthesis Alter the total population of active sites rather than influencing the efficiency of existing enzyme molecules

Enzymes that are needed at only one stage of development or under selected physiologic conditions are subject to regulation of synthesis

Enzymes that are in constant use are NOT regulated by altering the rate of enzyme synthesis

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Mechanisms for Regulating Enzyme Activity

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Regulator event

Typical effector

Results Time required for

changeSubstrate

AvailabilitySubstrate Change in

velocityImmediately

Product inhibition

Product Change in Vmax and/or Km

Immediately

Allosteric control

End product Change in Vmax and/or Km

Immediately

Covalent modification

Another enzyme Change in Vmax and/or Km

Immediately - minutes

Synthesis or degradation of

enzyme

Hormone or metabolite

Change in the amount of

enzyme

Hours to days

Enzyme Activity is Often Regulated

Feedback inhibition - a common form of enzyme regulation in which the product inhibits the enzyme .

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Enzymes - Activity Temperature and pH effect enzyme action

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Enzymes - Activity Temperature and pH effect enzyme action

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Enzymes - Activity Enzyme and substrate concentrations

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ENZYMES IN CLINICAL USE

Enzyme inhibitors as DRUGSEnzymes in CLINICAL DIAGNOSIS

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Enzyme inhibitors as DRUGS

1. STATINS – HMG Coenzyme A reductase inhibitors; lower serum lipid concentration

2. EMTRICTABINE and TENOFOVIR DISOPROXIL FUMARATE – inhibitors of viral reverse transcriptase; block replication of HIV

3. ACE Inhibitors (Captopril, Lisinopril, Enalapril) – antihypertensive agents

4. Lactam Antibiotics (Penicillin and Amoxicillin) – inhibitors of alanyl alanine carboxypeptidase-transpeptidase, thus blocking cell wall synthesis

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Enzymes in CLINICAL DIAGNOSIS

2 GROUPS OF PLASMA ENZYMES(1) Actively secreted into the plasma by

certain organs(2) Released from the cells during normal

cell turnover Intracellular, have no physiologic function

in the plasma Constant level in healthy individuals and

represent a steady state

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Elevated enzyme activity in the plasma may indicate tissue damage

accompanied by increased release of intracellular enzymes, thus useful as a

diagnostic tool

Elevated levels of ALT (alanine

aminotransferase; also called

glutamate: pyruvate transaminase; GPT)

signals damage

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ISOENZYMES

Also called isozymes Enzymes that catalyze the same reaction but

differ in their physical properties because of genetically determined differences in amino acid sequence

Different organs frequently contain characteristic proportions of different isoenzymes

Isoenzymes found in the plasma serve as a means of identifying the site of tissue damage

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CK, Creatinine kinase

also called Creatinine phosphokinase (CPK) 3 isoenzymes; CK1, CK2, and CK3 Each isoenzyme is a dimer composed of 2

polypeptides (B and M subunits: CK1=BB, CK2=MB, CK3=MM)

CK2(MB) isoenzyme is present in more than 5% in myocardial muscles

Appears approximately 4 to 8 hours following onset of chest pain, and reaches a peak in activity at approximately 24 hours

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LACTATE DEHYDROGENASE (LDH)

Elevated following an infarction peaking 3 to 6 days after the onset of symptoms

Of diagnostic value in patients admitted more than 48 hours after the infarction

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Principal Serum Enzymes Used in Clinical Diagnosis

Serum Enzyme Major Diagnostic Use

AminotransferasesAspartate aminotransferase (AST, or SGOT)Alanine aminotransferase (ALT, or SGPT)

Myocardial infarction

Viral hepatitis

Amylase Acute pancreatitis

Ceruplasmin Hepatolenticular degeneration (Wilson’s disease)

Creatinine kinase Muscle disorders and myocardial infarction 100

Principal Serum Enzymes Used in Clinical Diagnosis

Serum Enzyme Major Diagnostic Use

-Glutamyl transpeptidase Various liver diseases

Lactate dehydrogenase (isoenzymes)

Myocardial infarction

Lipase Acute pancreatitis

Phosphatase, acid

Phosphatase, alkaline

Metastatic carcinoma of the prostate

Various bone disorders, obstructive liver diseases

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enzymes 1. history of enzymes -1700s and early 1800s, the digestion of meat by stomach secretions...

Documents

enzyme structuremost

set of enzymes

enzyme inhibitors

enzyme action

term enzyme

living yeast cells

enzyme inhibitor inducer

high activity