enzymes

2

Enzymes

Dr

Mohamed Mostafa Omran

Faculty of Sciences, Helwan University

Introduction to Biochemistry

Biochemistry

• Chemistry of living organisms.

• Each second/ 8,000 trillion reactions in

our bodies

Biomolecules

• Carbohydrates

• Lipids

• Proteins

• Nucleic acid

• Vitamins

• Enzymes

Enzymes

Enzymes

Made of protein Present in

all living cells

Converts substrates

into products

Biological

catalysts

Increase the rate of

chemical reactions

proceeding from10^3–10^8 times

faster than uncatalyzed reactions.

Remain unchanged

by chemical reaction

Affected by cellular conditions: any condition that affects protein structure temperature, pH, salinity

Properties of enzymes

• Reaction specific

– each enzyme works with a specific substrate

• chemical fit between active site & substrate

– H bonds & ionic bonds

• Not consumed in reaction

– single enzyme molecule can catalyze thousands or more reactions per second

• enzymes unaffected by the reaction

• Affected by cellular conditions

– any condition that affects protein structure

• temperature, pH, salinity

Specificity of the Enzyme • For enzyme and substrate to react, surfaces of each must

be complementary

• Enzyme specificity: the ability of an enzyme to bind only one, or a very few, substrates thereby catalyzing only a single reaction

• Compare these 2 reactions:

• Urease is VERY

Specific or has a

HIGH DEGREE of

Specificity

Specificity of Enzymes One of the properties of enzymes that makes them so important

as diagnostic and research tools is the specificity they exhibit relative to the reactions

they catalyze.

In general, there are four distinct types of specificity:

1. Absolute specificity - the enzyme will catalyze only one reaction.

2. Group specificity - the enzyme will act only on molecules that have specific

functional groups, such as amino, phosphate and methyl groups.

3. Linkage specificity - the enzyme will act on a particular type of chemical bond

regardless of the rest of the molecular structure.

4. Stereochemical specificity - the enzyme will act on a particular steric or optical

isomer. Though enzymes exhibit great degrees of specificity, cofactors may serve many

apoenzymes.

Enzyme Specificity

Function of Enzymes

• Enzymes speed up the rate of chemical reactions in the body; both breaking down (e.g.: starch into maltose) and building up reactions. (e.g: amino acids into proteins).

• Enzymes lower the activation energy required to start a chemical reaction

Characteristics of Enzymes • Enzymes are highly specific

in action.

• Enzymes remain chemically unchanged at the end of the reaction.

• Enzymes are required in minute amounts.

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is a non-protein part firmly attached to the apoprotein

14

Cofactors are bound to the enzyme for it to maintain the correct configuration of the active site

How Coenzymes work

• A coenzyme is required by some enzymes

– An organic molecule bound to the enzyme by weak interactions / Hydrogen bonds

– Most coenzymes carry electrons or small groups

– Many have modified vitamins in their structure

Water-Soluble Vitamins and Their Coenzymes

Turnover number (Enzyme Unit)

The “turnover number” is the number of

substrate molecules converted into product

by an enzyme molecule in a unit time under

optimal condition of measurement.

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Zymogens (proenzymes)

• Digestive and coagulation enzymes are secreted in inactive

forms, zymogens or proenzymes.

• Zymogens are activated by trimming of a short peptide

blocking the active site, or by covalent modification of the

zymogen.

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Mechanism of activation

* The mechanism of activation may involve unmasking of a

polypeptide chain that may be blocking or masking the active

centers of apoenzyme.

* Proteolytic enzymes are secreted inactive to prevent

digestion of protein of the cell that synthesized them.

Activation takes place by:

HCL

1- pH – changes: Pepsinogen Pepsin

trypsin

2- Auto-activation: Trypsinogen Trypsin

Entrokinase

3- By other enzymes: Trypsinogen Trypsin

Protein ase Substrate Name + -ase

Carbohydrate ase

Terminology of enzymes

1-Some enzymes still retain their old names as digestion enzymes still use –in

pepsin, trypsin.

2-End in –ase:

Identifies a reacting substance

lipase - reacts lipid

3-Describes function of enzyme:

oxidase – catalyzes oxidation

hydrolase – catalyzes hydrolysis

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Terminology of enzymes

Nomenclature systems are

4. The trivial name: which give no indication of the function of the

enzyme, are commonly used.

In some cases, the trivial name is composed of the name of the substrate

involved, the type of the reaction catalyzed, and the ending – ase.

Lactate + dehydrogenation + ase = lactate dehydrogenase

5. The systematic name:

It is made up of the names of substrates acted upon , coenzyme involved in

the reaction , the type of reaction catalyzed+ ase

eg Lactate NAD+ Oxidoreductase (the old name was lactate

dehydrogenase).

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Classification of enzymes: six classes according to reaction type

(Each class comprises other subclasses)

Enzyme class General scheme of reaction

1. Oxidoreductases Ared + Box Aox + Bred

2. Transferases A-B + C A + C-B

3. Hydrolases A-B + H2O A-H + B-OH

4. Lyases A-B A + B (reverse reaction: synthases)

5. Isomerases A-B-C A-C-B

6. Ligases (synthetases) A + B + ATP A-B + ADP + Pi

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1 Oxidoreductases

catalyze the oxidation or reduction of substrate

subclasses:

1. dehydrogenases catalyze transfers of two hydrogen atoms

2. oxygenases catalyze the incorporation of one/two O atoms

into the substrate (monooxygenases, dioxygenases)

3. oxidases catalyze transfers of electrons between substrates (e.g.

cytochrome c oxidase, ferroxidase)

4. peroxidases catalyze the breakdown of peroxides

Example: lactate + NAD+ pyruvate + NADH + H+

lactate dehydrogenase

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2 Transferases

catalyze the transfer of a group from one to another substrate

subclasses:

• aminotransferases, methyltransferases,

• phosphomutases – the transfer of the group PO32– within molecule

• kinases phosphorylate substrate by the transfer of phosphoryl group

PO32– from ATP (e.g. hexokinases, proteinkinases)

Example: glucose + ATP glucose 6-P + ADP

glucokinase

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Example: glucose 6-P + H2O glucose + Pi

glucose 6-phosphatase

3 Hydrolases

catalyze the hydrolytic splitting of esters, glycosides, amides, peptides etc.

subclasses:

• esterases (lipases, phospholipases, ribonucleases, phosphatases)

• glycosidases (e.g. sucrase, maltase, lactase, amylase)

• proteinases and peptidases (pepsin, trypsin, cathepsins,

dipeptidases, carboxypeptidases, aminopeptidases)

• amidases (glutaminase, asparaginase)

• ATPases (split anhydride bonds of ATP)

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4 Lyases

catalyze non-hydrolytic splitting or forming bonds C–C, C–O, C–N, C–S through

removing or adding, respectively, a small molecule (H2O, CO2, NH3)

Some frequent recommended names:

• ammonia lyases (e.g. histidine ammonia lyase: histidine urocanate + NH3)

• decarboxylases (amino acid amine + CO2)

• aldolases (catalyze aldol cleavage and formation)

• (de)hydratases (carbonate dehydratase: CO2 + H2O H2CO3)

Example: fumarate + H2O L-malate

fumarate hydratase

Fumarate is hydrated to malate in a freely reversible reaction

catalyzed by fumarase (also called fumarate hydratase)

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6 Ligases

catalyze formation of high-energy bonds C–C, C–O, C–N

in the reactions coupled with hydrolysis of ATP

Frequent recommended names:

carboxylases

synthetases

(e.g. glutamine synthetase: glutamate + ATP + NH3 glutamine + ADP + Pi)

Example: pyruvate + CO2 + ATP + H2O oxaloacetate + ADP + Pi

pyruvate carboxylase

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Three enzymes have something to do with phosphate

Enzyme (Class) Reaction scheme / Reaction type

Kinase (Transferase)

substrate-OH + ATP substrate-O-P + ADP

phosphorylation = transfer of phosphoryl PO32– from ATP to substrate

Phosphatase (Hydrolase)

substrate-O-P + H2O substrate-OH + Pi

the hydrolysis of phosphoester bond

Phosphorylase (Transferase)

(glycogen)n + Pi (glycogen)n-1 + glucose 1-P

inosine + Pi hypoxanthine + ribose 1-P

phosphorolysis = the splitting of glycoside bond by

phosphate = transfer of glucosyl to inorganic phosphate

!

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Distinguish:

Three types of lysis (decomposition of substrate)

Hydrolysis

the decomposition of substrate by water, frequent in intestine:

sucrose + H2O glucose + fructose

(starch)n + H2O maltose + (starch)n-2

Phosphorolysis the cleavage of O/N-glycoside bond by phosphate:

(glycogen)n + Pi (glycogen)n-1 + glucose 1-P

Thiolysis

the cleavage of C-C bond by sulfur atom of coenzyme A

in β-oxidation of FA or ketone bodies catabolism

RCH2COCH2CO-SCoA + CoA-SH RCH2CO-SCoA + CH3CO-SCoA

!

0

35 Time Salt concentration

37°

Temperature

temperature

rea

ctio

n r

ate

Factors affecting enzyme

function • Temperature

– Optimum T°

• greatest number of molecular collisions

• human enzymes = 35°- 40°C

– body temp = 37°C

– Heat: increase beyond optimum T°

• increased energy level of molecules disrupts bonds in enzyme & between enzyme & substrate

– H, ionic = weak bonds

• denaturation = lose 3D shape (3° structure)

– Cold: decrease T°

• molecules move slower

• decrease collisions between enzyme & substrate

Enzymes and temperature • Different enzymes function in different

organisms in different environments

37°C temperature

rea

ctio

n r

ate

70°C

human enzyme hot spring bacteria enzyme

(158°F)

7

pH

pH

react

ion

rate

2 0 1 3 4 5 6 8 9 10

pepsin trypsin

11 12 13 14

pepsin

trypsin

Factors affecting enzyme function • pH

– changes in pH

• adds or remove H+

• disrupts bonds, disrupts 3D shape

– disrupts attractions between charged amino acids

– affect 2° & 3° structure

– denatures protein

– optimal pH?

• most human enzymes = pH 6-8

– depends on localized conditions

– pepsin (stomach) = pH 2-3

– trypsin (small intestines) = pH 8

7 2 0 1 3 4 5 6 8 9 10 11

Salinity

salt concentration

rea

ctio

n r

ate

Factors affecting enzyme function • Salt concentration

– changes in salinity

• adds or removes cations (+) & anions (–)

• disrupts bonds, disrupts 3D shape

– disrupts attractions between charged amino acids

– affect 2° & 3° structure

– denatures protein

– enzymes intolerant of extreme salinity

• Dead Sea is called dead for a reason!

Substrate Concentration

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As substrate concentration

increases, the rate of reaction

increases (at constant enzyme

concentration).

• the enzyme eventually

becomes saturated giving

maximum

activity.

Important conclusion about Km 1. Substrate are usually in physiological fluids in amounts nearly equal to Km values.

2. Km is a constant characteristic of an enzyme and its substrate. Km reflect the affinity

of the enzyme for the substrate.

3. Low Km reflect high affinity of the enzyme for substrate

4. High Km reflect low affinity of the enzyme for substrate

• Glucose Hexokinase or glucokinase Glucose 6 phosphatase

• Hexokinase is more active than glucokinase

• because the amount of glucose (substrate) needed to produce ½ V max in case of

hexokinaes is less than in case of glucokinase

• i.e Km of hexokinase is less than glucokinase.

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Uncompetitive

Classes of Inhibition

Two real, one hypothetical

• Competitive inhibition - inhibitor (I) binds only to E, not to ES

• Noncompetitive inhibition - inhibitor (I) binds either to E and/or

to ES

• Uncompetitive inhibition - inhibitor (I) binds only to ES, not to

E. This is a hypothetical case that has never been documented

for a real enzyme, but which makes a useful contrast to

competitive inhibition

Mechanisms for Regulating Enzyme Activity

1. Allosteric Enzymes

• Allosteric means "other site" or "other structure".

• Enzymes whose activity can be changed by molecules (effector

molecules) other than substrate.

• The interaction of an inhibitor at an allosteric site changes the

structure of the enzyme so that the active site is also changed.

2 Processes Involving the Allosteric Enzyme

1. Negative Allosterism: effector binding sites alters

the shape of the active site of the enzyme making

it to an inactive configuration.

2. Positive Allosterism

- effector binding sites that alters the shape

of inactive site of enzyme to an active

configuration.

Therefore, binding of the effector molecule

regulates enzyme activity by determining

whether it will be active or not.

Vo vs [S] for Allosteric Enzymes

2. Feedback Inhibition

• An enzyme regulation process in which formation of a

product inhibits an earlier reaction in the sequence. It

controls the allosteric enzymes.

3. Proenzymes (Zymogen) • The inactive form of enzyme which can be activated

by removing a small part on their polypeptide chain.

• Mostly are the digestive enzymes and blood clotting

enzymes.

• Why is it that digestive enzymes are in inactive state

before it becomes active?

• This is necessary to prevent digestion of pancreatic

and gastric tissues.

Zymogen

• Pepsinogen

• Trypsinogen

• Prothrombin

Active Form of Enzyme

• Pepsin

• Trypsin

• Thrombin

4. Phosphorylation:

• Some enzymes may be regulated by addition or

removal of phosphate groups from enzymes

• phosphate is transferred from an activated donor

(usually ATP) to an amino acid on the regulatory

enzyme, is the most common example of this type of

regulation.

Phosphorylation

• Phosphorylation reactions are catalyzed by protein kinase

and removed by protein phosphatase.

• The phosphorylated enzyme may be more or less active the

unphosphorylated enzymes.

• Phosphorylation of glycogen phosphorylase enzyme result

in increase enzyme activity.

• Phosphorylation of glycogen synthase enzyme result in

less enzyme activity.

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5. Isoenzymes: are enzymes catalyze the same chemical reaction

(identical functions), isolated from different tissues, have same

number but different amino acid sequence.

Differences may be:

– A.acid sequence

– Some covalent modification

– 3-D structure

– The existence of isozymes permits the fine-tuning of

metabolism to meet the particular needs of a given tissue or

developmental stage.

– Isoenzymes e.g

– Creatine kinase (CK)

– Creatine phosphokinase CPK

– alkaline phosphatase (ALP)

Percentage of Enzymes Usage in Industries:

Detergentes

• Contain:

- lipase: greasy stains

- protease: eggs, blood

• Advantage: they work at lower

temperatures, so less water heating is

needed, and clothes don´t shrink.

Food industry

• Fruit juices: are extracted using pectinase. It

breaks down pectin and is much easier to squeeze

juice from the fruit. It also makes the juice clear

rather than cloudy.

• Biscuits: - isomerase: converts glucose to

fructose, which is sweeter so less needs to be used

in slimmers biscuits.

- protease: softens glutens, making the

roller of biscuits easier

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Three utilizations of enzymes in medicine

1. enzymes as indicators of pathological

condition

2. enzymes as analytic reagents in clinical

chemistry

3. enzymes as drugs

Enzyme Disease

Serum acid phosphatase Cancer prostate

Serum Alkaline phosphatase *Metastasis in liver, jaundice due to

carcinoma head of pancreas, osteoblastic

metastasis in bones

*Metastasis, or metastatic disease, is the

spread of a cancer from one organ or part to

another non-adjacent organ or par

Serum LDH Advanced malignancies and Leukemias

Β- Glucuronidase Cancer of urinary bladder

Leucine Amino Peptidase (LAP) Liver cell carcinoma

Neuron specific Enolase Malignancies of nervous tissue and brain

Enzymes as tumor markers

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NAME OF THE ENZYME Conditions in which level of activity

in serum is elevated

Aspartate Amino transferase (AST)

Serum glutamate-oxaloacetate

transaminase (SGOT)

Myocardial infarction, Liver disease

especially with liver cell damage

Alanine Amino transferase (ALT)

Serum glutamate-pyruvate

transaminase (SGPT)

Liver disease especially with liver cell

damage

Alkaline Phosphatase (ALP) Liver disease- biliary obstruction

Osteoblastic bone disease-rickets

Acid Phosphatase (ACP) Prostatic carcinoma

glutamyl Transferase ( GT) Liver disorder like liver cirrhosis and

alcoholism

Creatine kinase (CK) Myocardial infarction and skeletal

muscle disease(muscular dystrophy

Lactate Dehydrogenase (LDH) Myocardial infarction, other diseases

like liver diseases, some blood

diseases

Amylase Acute pancreatitis

Summary-Enzymes as diagnostic markers

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Enzyme Used for testing

Urease Urea

Uricase Uric acid

Glucose oxidase Glucose

Cholesterol oxidase Cholesterol

Lipase Triglycerides

Alkaline phosphatase ELISA

Horse radish Peroxidase ELISA

Restriction endonuclease Recombinant DNA technology

Reverse transcriptase Polymerase chain reaction

Enzymes as diagnostic reagents

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Enzyme Therapeutic Application

Trypsin, lipase and amylase Pancreatic insufficiency

Asparaginase/Glutaminase Acute lymphoblastic leukemias

Hyaluronidase Enhanced local anesthesia and for easy

diffusion of fluids

Papain Anti inflammatory

Serratopeptidase Pain killer and Anti inflammatory

Chymotrypsin Pain killer and Anti inflammatory

Alpha- 1 Antitrypsin Deficiency and Emphysema

Enzymes as therapeutic agents

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enzymes

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