Enzymes

Biological Catalysts Nomenclature and Classification

Enzyme-Substrate Interaction
Effects of pH and Temperature Regulation of Enzyme Activity Cofactors and Coenzymes Vitamins and Coenzymes
Ch106; Chpt 21 Enzymes 20 - 1

Enzymes = Biological catalysts Large proteins

Permit reactions to go at body conditions pH 7.4, 37oC Process millions of molecules every second Very specific react with only 1 or a few types of molecules (substrates).
Ch106; Chpt 21 Enzymes 20 - 2

Effect of enzymes on Eact

For all reactions you must get over the activation energy hurdle.

Ea

Energy

Reactants H2O2 H Products H2O + O2

Ch106; Chpt 21 Enzymes

20 - 3

Effect of enzymes on Eact

Enzyme catalyzed reaction

Enzymes change how reactions proceed.

Ea

Reducing activation energy.

Makes faster.
Products H2O + O2

Energy

Reactants H2O2 H

Ch106; Chpt 21 Enzymes

20 - 4

Enzyme nomenclature
Name is based on:
what with or how it reacts Examples To react with lactose.

-ase ending

lactase
To remove carboxyl from pyruvate.

pyruvate decarboxylase
Ch106; Chpt 21 Enzymes 20 - 5

Classification of enzymes
Based on type of reaction

Oxireductase catalyze a redox reaction Transferase Hydrolase Lyase Isomerases Ligase

Ch106; Chpt 21 Enzymes

transfer a functional group catalyze hydrolysis rxns Add or remove to C=C bonds rearrange to form isomers join two molecules
20 - 6

The Active Site

Enzymes are typically HUGE proteins, yet only a small part are actually involved in reaction. The active site has two basic components.

catalytic site

binding site
Model of trios-p-isomerase

Ch106; Chpt 21 Enzymes

20 - 7

The Active Site

Catalytic site
Binding Site
holds substrate in place Where reaction occurs

Substrate

Enzyme
Ch106; Chpt 21 Enzymes 20 - 8

SPECIFICITY
Enzymes are very specific. Each enzyme will catalyze only one type of reactions and often will only work with a specific substrate. Ex. NH2-C-NH2 + H20
urease

2NH3 + CO2

O
Urease has no effect on other compounds. Such absolute specificity is rather rare among enzymes.

Ch106; Chpt 21 Enzymes

20 - 9

Enzyme classes
Absolutely specific Only reacts with a single substrate. Group specific Works with similar molecules with the same functional group. Linkage specific Catalyzes a specific combination of bonds. Stereochemically specific Only will work with the proper D- or L- form.
Ch106; Chpt 21 Enzymes 20 - 10

 ISOENZYMES: Different enzymes that perform the same type of function in different organisms or tissues.

Ch106; Chpt 21 Enzymes

20 - 11

Enzyme-substrate complex
Step 1: (All of these steps are in equilibrium) Enzyme and substrate combine to form complex E + S ES Enzyme Substrate Complex

Ch106; Chpt 21 Enzymes

20 - 12

Enzyme-product complex
Step 2: An enzyme-product complex is formed.

ES

EP

ES

transition state

EP

Ch106; Chpt 21 Enzymes

20 - 13

Product
The enzyme and product separate

EP

E + P
The product is made Enzyme is ready for another substrate.

EP

Ch106; Chpt 21 Enzymes

20 - 14

Ch106; Chpt 21 Enzymes

20 - 15

Lock and Key Theory

Enzyme is lock and Substrate is the key. Substrate structure must fit into enzymes structure.

Ch106; Chpt 21 Enzymes

20 - 16

Induced Fit Theory

Active site may not fit substrate.

Site must change in order to form the complex.

Ch106; Chpt 21 Enzymes

20 - 17

Ch106; Chpt 21 Enzymes

20 - 18

Effect of Temp on Enzymatic Rxns

Exceeding normal pH and temperature ranges always reduces enzyme reaction rates.

Reaction Rate

Optimum Temp usually 37oC.

Temperature
Ch106; Chpt 21 Enzymes 20 - 19

Effect of pH on Enzymatic Rxns

Reaction Rate

Most enzymes work best near pH 7.4 not all though.

pH
Ch106; Chpt 21 Enzymes 20 - 20

Examples of optimum pH
Enzyme pepsin sucrase catalase arginase alkaline phosphatase Source gastric mucosa intestine liver beef liver bone Optimum pH 1.5 6.2 7.3 9.0 9.5

Ch106; Chpt 21 Enzymes

20 - 21

Effect of substrate concentration

For non-enzyme catalyzed reactions
Rate of reaction (velocity)

Rate increases if concentration of the substrate increases Substrate concentration

Ch106; Chpt 21 Enzymes

20 - 22

Effect of substrate concentration

For Enzyme catalyzed reactions Rates increase but only to a certain point Saturation point Vmax w/ more enzyme
Vmax w/ some enzyme At Vmax the enzyme is working as fast as it can. Rate is limited by the concentration of both the substrate and enzyme. Substrate concentration
Ch106; Chpt 21 Enzymes 20 - 23

Rate of reaction (velocity)

Effect of Enzyme concentration

Enzyme Activity

Enzyme Concentration

Ch106; Chpt 21 Enzymes

20 - 24

Turnover Number
Turnover Number:
The rate at which an enzyme transforms the substrate Is measured at optimum pH and temperature. Example:

Carbonic Anhydrase
H2CO3 H2O + CO2

36,000,000 molecules minute

Ch106; Chpt 21 Enzymes 20 - 25

ENZYME INHIBITION
Inhibitors = interfere with ability of enzyme to react properly with its substrate.
For example: Medicinal drugs inhibit by inactivating an enzyme essential to bacterial growth. Viruses more difficult to inhibit because they use enzyme system of the host cell. (An inhibitor of a virus also destroys host cells)
Ch106; Chpt 21 Enzymes 20 - 26

ENZYME INHIBITION
Two Types of Inhibitors: Competitive Noncompetitive

Ch106; Chpt 21 Enzymes

20 - 27

COMPETITIVE INHIBITOR

Competes with substrate for the active site.

Enzyme mistakes inhibitor for substrate

Ch106; Chpt 21 Enzymes 20 - 28

Reversible Competitive inhibition

Enzyme - substrate reactions in equilibrium.

Inhibitor

Substrate

EI

ES

EI
shifts

I + E + S Inc I Inc S

ES
Shifts

EP E + P
20 - 30

Ch106; Chpt 21 Enzymes

Competitive Inhibitors Sulfa Drugs

Illnesses caused by invading microorganisms like bacterium can be combated using a competitive inhibitor called an antimetabolite.

Folic Acid is a coenzyme in many biosynthetic processes like synthesis of amino acids and nucleotides.

Ch106; Chpt 21 Enzymes

20 - 31

Sulfa Drugs
Folic Acid : obtained from the diet or
H N H

O C OH

p-aminobenzoic acid

from microorganisms in the intestinal tract.

Microorganisms make folic acid from PABA.
H2N N OH N N N NH O CH2 CH2 C OH O
Ch106; Chpt 21 Enzymes 20 - 32

C NH CH C OH

Ch106; Chpt 21 Enzymes

20 - 34

Penicillin: War of Enzyme against Enzyme.

Produced by mold, it prevents growth of bacteria by successfully competing for active sites on an enzyme that bacteria need for cell wall production. 1. Bacteria need the enzyme transpeptidase to make their cell walls rigid and cross-linked. 2. Penicillin takes control of transpeptidase. 3. Bacteria cell walls are not cross-linked and the contents of the bacteria cells cannot be held in. 4. Cytoplasm spills out, and the bacteria die.
Ch106; Chpt 21 Enzymes 20 - 36

By changing the R group, science has found a way to prevent this from happening.
Ch106; Chpt 21 Enzymes 20 - 38

Non competitive Inhibition

This type of inhibitor is believed to alter the shape of the enzyme and greatly reduce its affinity for the substrate. 1. It does not compete with the substrate for the active site. 2. It does not need to resemble the structure of the substrate. 3. Its effect cannot be reversed by increasing the substrate concentration.

Ch106; Chpt 21 Enzymes

20 - 39

Non competitive Inhibition

A noncompetitive inhibitor can bind to an enzyme in many ways. If it binds somewhere on the surface of the enzyme, it causes a change in the tertiary structure. The substrate is inhibited because it cant get into the enzyme.

Ch106; Chpt 21 Enzymes

20 - 40

Regulation of enzyme activity

Enzymes are often regulated by the cell. (Unlike other catalysts) Cells use several methods to control when & how well enzymes work.

Ch106; Chpt 21 Enzymes

20 - 41

PROENZYMES (ZYMOGENS)
Enzymes manufactured in inactive form.

Activated when small part of polypeptide chain removed.

Hormones, Digestive Enz, Blood Clotting Enz

Ch106; Chpt 21 Enzymes 20 - 42

PROENZYMES (ZYMOGENS)
Enzymes manufactured in inactive form.
In pancreas (inactive) In blood (active)

Proinsulin
S
S

Insulin
S

S S

S S

Ch106; Chpt 21 Enzymes

20 - 43

PROENZYMES (ZYMOGENS)
(inactive) In pancreas Trypsinogen
enteropeptidase

(active) In Intestines Trypsin

Chymotrypsinogen

Trypsin

Chymotrypsin

procarboxypeptidase Trypsin Carboxypeptidase Digestive Enzymes

Ch106; Chpt 21 Enzymes

Proteases
Cleave peptides
20 - 44

PROENZYMES (ZYMOGENS)
(inactive) In pancreas Trypsinogen
enteropeptidase

(active) In Intestines Trypsin

Chymotrypsinogen

Trypsin

Chymotrypsin

procarboxypeptidase Trypsin Carboxypeptidase Activation in pancreas rather than intestines pancreas proteins get digested pancreatitis (inflammation of pancreas).
Ch106; Chpt 21 Enzymes 20 - 45

PROENZYMES (ZYMOGENS)
(inactive) In Gastric mucosa (active) In Stomach

Pepsinogen

H+

Pepsin

Digestive Enzyme

HCl
Produced as Food enters stomach

As pH acid Proenzyme gets cleaved Pepsin gets activated

Ch106; Chpt 21 Enzymes 20 - 46

Allosteric Enzymes
Application of non competitive inhibition Regulates away from active site Inactive Enzyme Active Enzyme Substrate Now fits

Positive Active Site Regulator Changed Positive allosterism - activates the enzyme. Negative allosterism - deactivates the enzyme.
Ch106; Chpt 21 Enzymes 20 - 49

Inactive Enzyme E1

Negative Regulator

Feedback Control
End Product Stops E1

B A

E1 Active Allosteric Enzyme

E1

E2

E3

Ch106; Chpt 21 Enzymes

20 - 50

Apoenzyme
protein portion Inactive

Cofactors

Co2+

Cofactor Non protein Group need to activate apoenzyme

Ch106; Chpt 21 Enzymes

Co2+

20 - 54

Mineral Cofactors
Metal Ion Cu2+ Fe2+/Fe3+ Enzyme involved Cytochrome oxidase Catalase Cytochrome oxidase Function redox redox

Zn2+

Alcohol dehydrogenase Used with NAD+

Mg2+

Glucose-9-phosphatase Hydrolyzes phosphate esters

Ch106; Chpt 21 Enzymes

20 - 56

Coenzymes
Organic molecule that temporarily binds to

apoenzyme in order for it to work

apoenzyme Protein
Ch106; Chpt 21 Enzymes

coenzyme
Non-Protein

holoenzyme
Total
20 - 57

Vitamins are often converted to coenzymes

Vitamin B1 B2 folic acid Coenzyme made Function

thiamine pyrophosphate decarboxylation flavin mononucleotide tetrahydrofolic acid carries hydrogen amino acid metabolism

biotin

biocytin

CO2 fixation
acyl group carrier

pantothenic Coenzyme A acid

Ch106; Chpt 21 Enzymes

20 - 59

Enzymes in Medical Diagnosis and Treatment

Most enzymes are confined within the cells of the body. However, small amounts can also be found in body fluids (blood, urine, cerebrospinal fluid) The level of enzyme activity outside the cells can be easily monitored. Abnormal activity (high or low) of particular enzymes in various body fluids signals either the onset of certain diseases or their progression.

Ch106; Chpt 21 Enzymes

20 - 60

Ch106; Chpt 21 Enzymes

20 - 61

EXAMPLES
Dead heart muscle cells spill their enzyme contents into the serum. The level of glutamate oxaloacetate transaminase (GOT) in the serum rises rapidly after a heart attack. The levels of GOT as well as lactate dehydrogenase and creatine phosphokinase are closely monitored in order to diagnose the severity of a myocardial infarction.
Ch106; Chpt 21 Enzymes 20 - 62

Specific enzyme examples

Lets look at role of some specific enzymes. Two good examples are: Chymotrypsin - A proteolytic enzyme (protein-cleaving). - Used in digestion of dietary protein in the small intestines. Acetylcholinesterase - Used for hydrolysis of acetylcholine. - Needed for operation of nerves.
Ch106; Chpt 21 Enzymes 20 - 63

Chymotrypsin
This enzyme is a proteolytic enzyme. It cleaves peptide bonds.
H O H | || | -C-HN - C - C - N - C | | | R H R1 Peptide bond

This enzyme only works on amino acids containing an aromatic ring. phenylalanine, tyrosine and tryptophan.
20 - 64

Ch106; Chpt 21 Enzymes

Acetylcholinesterase and nerve transmission

This enzyme is needed to transmit a nerve signal at a neuromuscular junction. Arrival of a nerve signal causes Ca2+ levels to increase.

This causes acetylcholine containing vesicles to move to end of the nerve cell where it is released.
Acetylcholine then diffuses across synapse to pass the signal to the muscle. Acetylcholinesterase then destroys the acetylcholine to stop the signal.
Ch106; Chpt 21 Enzymes 20 - 65

Acetylcholinesterase and nerve transmission

Presence of acetylcholine at receptor causes a flow of sodium and potassium ions. This causes a muscle contraction. acetylcholine receptor protein acetylcholinesterase - destroys excess acetylcholine
Ch106; Chpt 21 Enzymes 20 - 66

synaptic cleft

Acetylcholinesterase
Stick model of acetylcholinesterase.

Ch106; Chpt 21 Enzymes

20 - 67

Acetylcholinesterase and nerve transmission

Without the enzyme, muscles would continue to contract causing spasms. Acetylcholinesterase inhibitors are used as drugs and poisons. Organo fluorophosphates - bind to the enzyme. Death can occur. Succinylcholine Acts like acetylcholine and binds to sites on the muscle. Used as a muscle relaxant.
Ch106; Chpt 21 Enzymes 20 - 68

Another example
Blood Clotting - formation of fibrin. Process requires a series of enzymatic steps.

Many of the enzymes are made in an inactive form. This prevents blood from clotting on its own. Two pathways can be used to start the process. Extrinsic - Activated by tissue damage, outside the blood vessel. Intrinsic - Activated by damage within a blood vessel.
20 - 69

Ch106; Chpt 21 Enzymes

Fibrin

Ribbon model of fibrin.

Ch106; Chpt 21 Enzymes

20 - 71

Drug interactions
Drugs can be administered to alter the clotting mechanism. Example: Heparin - an anticoagulant. Acts by accelerating the action of the existing inhibitor of thrombin - antithrombin III. Antithrombin III inhibits activation of the clotting factors that have a reactive serine residue at their enzymatically active centers.
Ch106; Chpt 21 Enzymes 20 - 72

thrombin
Ch106; Chpt 21 Enzymes

antithrombin
20 - 73

Heparin interaction
thrombin antithrombin inhibited thrombin

serine

lysine sites heparin

Addition of heparin makes it easier for trombin to interact with antithrombin - positive allosteric effect.
Ch106; Chpt 21 Enzymes 20 - 74

Defective enzymes and disease

A number of hereditary diseases result from the absence of an enzyme or a defective one.
Disease Albinism Glactosemia Phenylketonuria (PKU) Tay-Sachs disease
Ch106; Chpt 21 Enzymes

Defective enzyme tyrosinase glactose 1-phosphate uridyltransferase phenylalanine hydroxylase hexosaminidase A

20 - 75

Phenylketonuria (PKU)
Genetic mutation that results in a defect of the enzyme phenylalanine hydroxylase. (carried by 2% of population) Affects about 1 baby per 13,000. Feds may require screening at birth. Can result in retarded physical and mental development if untreated. Treatment - restrict phenylalanine until age 10 (until brain is developed).
Ch106; Chpt 21 Enzymes 20 - 76

Phenylketonuria (PKU)
PKU is one of a family of enzymatic/genetic disorders related to phenylalanine metabolism.
phenylalanine -CH2-CH-COOH | NH2 tyrosine

PKU
blocked

HO-

-CH2-CH-COOH | NH2 albinism

alkaptonuria O CH-COO|| || + blocked CH C-SCoA 3 HC-COOacetyl CoA fumarate

Ch106; Chpt 21 Enzymes

melanin
-CH2-COOH Homogentisic acid
20 - 77

ALKAPTONURIA AND OCHRONOSIS

Alkaptonuria is a rare disease in which the body does not have enough of an enzyme called homogentisic acid oxidase (HGAO) homogentisic acid (HGA) is not used and builds up in the body Some is eliminated in the urine, and the rest is deposited in body tissues where it is toxic. The result is ochronosis, a blue-black discoloration of connective tissue including bone, cartilage, and skin caused by deposits of ochre-colored pigment.
Ch106; Chpt 21 Enzymes 20 - 78