EnzymesFor Medical Students

Dr Mustafa Younis

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Thousands of chemical reaction reactions proceeding

very rapidly at any given time within living cells

Transformation are catalyzed by enzymes which are

usually protein in nature.

Enzymes are biocatalysts accelerating the rate of

chemical reactions.

Enzyme catalysis is very rapid, one molecule of

enzyme can act on 1000moles of substrate/min.

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Lack of enzymes lead to block in the metabolicpathways causing a group of diseases termed inbornerrors of metabolism.

Definitions and terminology

Apoenzyme; is the protein part of the enzyme.

Coenzyme; is a nonprotein, low Mwt, heat stable substancebinding loosely with the enzyme and regenerated after thereaction (few coenzyme can bind firmly “covalently” and theyare termed prosthetic group).

Holoenzyme: represents the enzyme and its coenzyme.

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Substrate: the molecule upon which the enzyme act to

form the product.

Substrate binding site (active site): particular region

on the surface having a specific arrangement of

chemical groups formulated to bind a specific substrate.

Allosteric sites: some enzymes contain other sites

“allosteric sites” where small molecules (allosteric

effectors) can bind resulting in increased or decreased

activity of the enzyme for its substrate.

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Intracellular Location

of enzymes :presented the metabolic

pathway in each cell

compartment.

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Nomenclature14

Classification of enzymes

1. Oxidoreductases: catalyze oxidation and reduction reactions. Use oxygen as an electron acceptor but do not incorporate it into the substrate.

Examples:

Dehydrogenases: Use molecules other than oxygen (e.g, NAD+ ) as an electron acceptor.

Oxygenases: directly incorporate oxygen into the substrate.

Peroxidases: use H2O2 (hydrogen peroxide) as an electron acceptor.

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2. Transferase: transfer groups other than O2 and H.

Methyltransferases: transfer methyl groups between substrates.

Aminotransferases: transfer NH2 from amino acids to keto acids,

Kinases: transfer PO3− from ATP to substrate, e.g., Hexokinase:

Phosphorylases: transfer PO3− from inorganic phosphate to substrate.

Hexokinase

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3.Hydrolases: Hydrolyse in the presence of water.

Phosphatases: remove PO3− from substrate.

Phosphodiesterases: cleave phosphodiester bonds such as those in

nucleic acid.

Proteases: Cleave amide bonds such as those in proteins.

4. Lyases: cleaves C-C , C-S or certain C-N bonds without addition of

water. Some call it synthase (form new product without using ATP).

Decarboxylases: produce CO2 via elimination reaction.

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5.Isomerase: interconvert isomers, for example:

Racemases: interconvert L (levorotatary) and D (dextrorotatary)

stereoisomers.

Mutases: transfer groups between atoms.

6.Ligases: catalyze formation of bonds between C and O, S, N

coupled to hydrolysis of high energy phosphate (ATP).

Carboxylase: add CO2 to substrate.

Synthetases: link 2molecules via an ATP-dependent reaction.

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Catalytic activity

Substrate specificity and the active site:An enzyme catalyzed reaction is initiated when

the enzyme binds to form an enzyme-substratecomplex .

In general enzyme molecules are larger thansubstrate molecules.

Binding occurs at the active site of the enzyme.

The unique catalytic properties of the enzymeare based on its 3-dimensional structure and onthe active site whose chemical groups may bebrought into close proximity from differentregions of the polypeptide chain.

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Active site of enzyme1. Active site occupies a small part of the enzyme and is situated in a cleft

in the enzyme where the substrate binds.

2. Binding of the substrate to the active site depends on presence of specific groups or atoms in the active site for the substrate binding and catalysis.

3. During binding, these specific groups may realign themselves to provide the unique conformation permitting exact fitting of the substrate in the active site.

4. Binding of substrate to the active site is through non-covalent bonds (electrostatic bonds, hydrogen bonds and hydrophobic interactions).

5. Amino acid residues at the active site are called catalytic residues and catalysis occurs at this site.

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Models are postulated to explain substrate binding to the enzyme:

First Model:

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Second Model: Induced Fit Model:

The binding site is not fully formed. Binding of the substrate to the

enzyme will induce a conformational change in the enzyme directing

appropriate amino acids to the active site. Some times, these changes

are accompanied by changes in the substrate to provide a perfect fit

for substrate binding and catalysis.

Induced Fit Model

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Specificity of EnzymesEnzymes are highly specific and catalyze only one type ofreaction.

1. Absolute specificity: The enzyme is specific for onesubstrate e.g.; urease acts only on urea; glucose oxidaseoxidizes only glucose but not other monosaccharides.

2. Relative specificity: The enzyme acts on a group of closelyrelated substrates: pancreatic lipase hydrolyzes alpha esterbonds in triglycerides irrespective of the nature of fatty acidattached. (bond specificity).

3. Group specificity: Most proteolytic enzymes show groupspecificity, for example; trypsin hydrolyzes peptide bondsprovided only by arginine and lysine. (bond and groupspecificity).

4. Stereospecificity: Human enzymes are specific for L-aminoacids and D-monosaccharides.

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Activation Energy

Imagine a chemical reaction

as the process of rolling a huge

stone (reactant) up a hill so

that it rolls down and breaks

into tiny pieces (products).

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Activation energy is the

energy needed to roll the stone

up the hill.

Activation Energy

Imagine a chemical reaction

as the process of rolling a huge

stone (reactant) up a hill so

that it rolls down and breaks

into tiny pieces (products).

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Once over the hill, the rest of

the reaction occurs.

Activation Energy

Imagine a chemical reaction

as the process of rolling a huge

stone (reactant) up a hill so

that it rolls down and breaks

into tiny pieces (products).

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Activation energy is the

energy needed to roll the stone

up the hill.

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Activation Energy

Imagine a chemical reaction

as the process of rolling a huge

stone (reactant) up a hill so

that it rolls down and breaks

into tiny pieces (products).

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Activation energy is the

energy needed to roll the stone

up the hill.

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Once over the hill, the rest of

the reaction occurs.3

The stone rolls down and breaks into

tiny pieces (products are formed).4

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The stone rolls down and breaks into

tiny pieces (products are formed).

The energy needed to start a chemical

reaction is called activation energy.

Activation Energy

Imagine a chemical reaction

as the process of rolling a huge

stone (reactant) up a hill so

that it rolls down and breaks

into tiny pieces (products).

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Activation energy is the

energy needed to roll the stone

up the hill.

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Once over the hill, the rest of

the reaction occurs.3

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Enzymes as catalysts Enzymes lower the activation

energy of a reaction so that it occurs more readily.25

Factors affecting enzyme activity

1) Temperature.

2) pH

3) Substrate concentration.

4) Enzyme concentration.

5) Coenzymes and cofactors.

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Most enzymes pH 6-

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Blood pH 7.4

Pepsin pH 2

Acid phosphatase 4-5

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Features of coenzymes

i. Coenzymes are heat stable and mostly derived from vitamins.

ii. They are low molecular weight substances.

iii. The coenzyme combines loosely to the enzyme by non-covalent

linkage. When the reaction completed, the coenzyme is released

from the apoenzyme.

Coenzymes derived from water-soluble vitamins (B-complex group)

can be divided into 2 groups:

a) Coenzymes involved in hydrogen transfer reactions: they donate

or accept hydrogen or electrons, e.g., NAD+, NADP+ , FAD+ and.

Lactate + NAD+ Pyruvate + NAD+ + H +

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b) Coenzymes taking part in reactions transferring a group other than H + :

CO2 Biotin.

NH2 Pyridoxal phosphate (PLP).

Effect of Metals:

Metal cofactors may bound reversibly or tightly to enzymes.

Reversible binding occurs in Metal activated enzymes (e.g. magnesium in kinases and phosphotransferase).

Tight binding occurs in metalloenzymes (e.g. Ca2+ for lipases).

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Vmax

Vmax /2

Vmax /2

A

B

C

Vi

Substrate conc.[ S ]Km

Velocity (V)

Effect of substrate concentration

The initial rate ( or initial velocity, Vi) of an enzyme catalyzed reaction

is dependent on substrate concentration [ S ]. If substrate concentration

[ S ] increased, while other conditions kept constant, the initial

velocity, Vi “point A” (velocity measured when very little substrate has

Vmax /2

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Reacted) increased to a maximum velocityVmax (point C) with no subsequent increase.

Plotting the velocity of an enzyme catalyzed reaction at different substrate concentration is demonstrated in the figure:

Point A represents the initial velocity; small number of enzymes is occupied with the substrate [ES].

Point C represent maximal velocity (Vmax ); all free enzymes are saturated with the substrate and present as [ES].

Point B half of the enzyme molecules are saturated with the substrate, velocity is half maximal velocity (Vmax /2) at this enzyme concentration. The substrate concentration required to produce half maximal velocity of the enzyme catalyzed reaction is termed Michaelis constant “Km ”.

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Michaelis-Menten kinetic theory of enzyme action

Michaelis-Menten formulated an equation which relates the rate of

enzyme (velocity) catalyzed reaction to substrate concentration:

Where;

Vi –is the rate (or velocity) of the reaction.

Vmax –is the rate when the enzyme is fully saturated with substrate.

Km - Michaelis constant; is the substrate concentration at

which the reaction rate is half maximal velocity.

Vmax /2 [ S ]

Km + [ S ]Vi =

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Lineweaver-Burk Plot

Represents a linear form of Michaelis-Menten equation and it requires

few points to define Km ( it is the method often used to determine Km

which is expressed as molarity or moles/L).

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Importance of Km

1- Km is a constant for a particular enzyme under standardized

conditions.

2- Km values are used practically in enzyme assay.

3- Km and Vmax can be affected by pH, temperature and other factors.

4- Km denotes enzyme affinity for its substrate. The higher the

Km the lower the enzyme affinity for its substrate.

5- Km permits evaluation of the inhibitor type (explained later).

6- Isoenzymes differ in their Km .

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Enzyme Inhibition

Enzyme inhibition is one way of regulating enzyme activity. Most

therapeutic drugs function by inhibition of a specific enzyme.

In the body some of the processes controlled by enzyme inhibition

are blood coagulation, blood clot dissolution (fibrinolysis) and

inflammatory reactions.

Types of inhibitors:

1. Competitive inhibition.

2. Non-competitive inhibition.

3. Uncompetitive inhibition.

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Competitive inhibition:

Occurs at the substrate binding site.

The inhibitor is a structural analogue of the substrate, so both are competing for binding at the enzyme active site.

Succinate and malonate are 2 structural analogues. So malonate blocks the action of succinate dehydrogenase on succinate.

Allopurinol is a competitive inhibitor for xanthine oxidase and used to treat Gout.

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Example: Lead---forms

covalent bond with

sulfhydryl gp of cysteine in

protein

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C. Uncompetitive inhibitors:

In this case the inhibitor have no affinity for free

enzyme.

The inhibitor (I) bind to [ES] complex ESI

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Enzyme regulation

1-regulation of enzyme quantity:

The amount of enzyme may be

increased by increasing the rate of synthesis.

Decreased by decreasing the rate of degradation.

A- Regulation by induction

Induction of synthesis of a particular enzyme.

The effector is called inducer (substrate, Hormone).

B-Regulation by repression:

Number of enzyme molecules decreased by

repression.

The effector is called repressor.

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2-Regulation of catalytic activity

Regulatory enzymes:

Control of metabolic pathway may be accomplished by modulation of

key enzymes---catalyse the first step of metabolic sequence (rate-

limiting step) that control the overall pathway.

A-Allosteric regulation:

Regulate key enzyme.

Allosteric enzymes oligomeric proteins (more than one subunit).

Allosteric enzymes posses 2 sites:

i-Catalytic site (active site).

ii- allosteric site-where allosteric modifier bind.

Binding causes conformational changes in the enzyme which can

either increase (positive allosteric modifier) or decrease (negative

allosteric modifier).

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Feedback inhibition:

The end product of metabolic pathway results in

allosteric inhibition of the first enzyme in the pathway.

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B-Covalent Modification

Covalent modification ----Addition of group to the enzyme by

covalent bond or removal of group by cleaving the covalent

bond.

Covalent modification include phosphorylation and

dephosphorylation, acetylation and deacetylation, methylation

and demethylation.

In mammals phosphorylation and dephosphorylation.

Phosphoylation (OH group of -----Kinases(ATP).

Dephosphorylation-----Phosphoprotein phosphatase.

Glyogen synthase +P inactive (Glycogen

synthesis)

Glycogen phosphorylase+P active (Glycogen

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C-Proenzymes:

o Is another form of covalent modification but is irreversible.

o Some enzymes are synthesized and secreted in the form of

inactive precursor called zymogen.

o Zymogen Proteolytic cleavage active E + small polypeptide.

o Proteolytic cleavage conformational change reveals

active site.

1- Many proteolytic enzymes of the stomach and pancreas are

secreted as zymogens activated in alimentary canal (this

prevents autolysis of cellular structural proteins). Examples:

Pepsinogen, procarboxypeptidase and trypsinogen.

2- Enzymes of blood clot formation and dissolution secreted

as zymogens and activated when required.

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Hormonal regulation of enzymes:

Regulation via cAMP (cyclic adenosine

monophosphate) which is the second messenger of

may hormones (hormone is the first messenger).

cAMP activate protein kinases

phosphorylate

Target Enzymes

become

active or inactive

(covalent

modification)

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Enzymes in clinical medicine

The principles of enzymology outlined previously are

applied clinically in 3 ways:

1) Diagnosis and prognosis of diseases.

2) Some enzymes are used as therapeutic agents

3) Enzymes as diagnostic reagents.

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Diagnosis and prognosis of diseases

Changes in concentration and activity of plasma enzymes reflect

changes that have occurred in a particular tissue or organ.

Plasma enzymes are of two types:

1-Functional enzymes: synthesized in the liver and present in

the blood in high concentration (perform physiological functions

in the blood-----enzymes associated with blood coagulation..

2-Non-functional plasma enzymes:

intracellular enzymes present in very low levels in the

blood (in healthy state) and has no function.

They are released in the plasma as a result of cellular

damage (e.g myocardial infarction &hepatitis).

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Enzymes of Diagnostic importance

Enzyme Diagnostic Use

1. Amylase and Lipase

2. Acid phosphosphatase

3. Creatine kinase

4. Aspartate transaminase

(AST)

5. Alanine

aminotransferase

6. Alkaline phosphatase

Pancreatitis

Prostate cancer

Myocardial infarction and

Muscle diseases

Myocardial infarction and

hepatitis

Viral hepatitis

Bone & Liver diseases

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Isoenzymes in diagnosis

Isoenzymes are different molecular forms of the

same enzyme (differ in amino acid sequence).

Synthesized by different tissues.

Isoenzymes catalyze the same reaction.

They migrate differently in electrophoresis

(because they contain different numbers of charged

amino acids).

They are made of different subunits.

Isoenzymes of clinical application include: Lactate

dehydrogenase (LDH) and Creatine Kinase (CK).

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Lactate dehydrogenase (LDH):

Tetrameric enzyme formed by combination of 2 subunits: H

(Heart) and M (Muscle):

Total LDH is increased in hepatocellular damage, leukemia and

hemolytic anemia In Myocardial infarction total LDH as well as

LDH-1 increased.

Type Subunit Tissue of

origin

LDH-1

LDH-2

LDH-3

LDH-4

LDH-5

H4

H3M1

H2M2

H1M3

M4

Heart muscle

RBCs

Brain

Liver

Muscles

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CK is a dimer having 2 subunits : B for brain and M for

muscles:

CK-1---BB-brain, CK-2---MB-heart and CK-3---MM-

muscles.

In myocardial infarction (MI): both CK and LDH

increased

CK increased within 4-6hrs after chest pain and return to

normal within 3days.

LDH return to normal within prolonged time.

CK is used for early detection of MI and LDH for follow up

Creatine Kinase (CK)62

Enzymes as therapeutic agents

1. Streptokinase: prepared from streptococcus and used in

clearing blood clots in MI. Sterptokinase activates

plasminogen forming plasmin cleaves fibrin into

several soluble components.

2. Asparaginase: used in adult leukemia. Decreases

asparagine level which is needed for tumor cells.

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Enzymes as diagnostic reagents.

Determination of

Glucose oxidase glucose estimation

Uricase uric acid

Urease Urea

Cholesterol oxidase cholesterol

Lipase triglycerides

Enzymes used in ELISA ---technique used.

-THE END-

Thanks

Dr Mustafa

Younis