Enzymes - biological catalysts that speed up the metabolic reactions that occur in the body -very specific in action

in general, an enzyme catalyzes only one specific reaction Ex: amylase (found in the human digestive tract) catalyses only the hydrolysis of starch to yield glucose. Amylase can not act on the hydrolysis of cellulose and others - the substrate for this reaction is the starch Substrate the compound that the enzyme catalyzes

Holoenzyme - enzyme that needs other molecule in order to function - made up of the cofactor + the apoenzyme - neither the cofactor or apoenzyme is active apoenzyme - protein component

cofactor - nonprotein component

ex. of cofactors are inorganic ions such as Mg2+ and Zn2+ and vitamins (also called coenzymes) coenzymes - substances that transport substrates from one enzyme to another

Enzyme action - most enzymes are globular proteins - the enzymes shape enables it to receive only one type of molecule that molecule that will fit into its shape. this makes the enzyme highly specific - the place where the substance fits into the enzyme is called the active site - an the substance that fits into the active site is called the substrate - when the substrate joins the enzyme, the entire structure is called the enzyme-substrate complex - the complex leads to the formation of the transition-state species - the substrate becomes changed by the enzymes action and is then released as the product. - the enzyme is then free to join another substrate -

Models of enzyme action

- there is a particular site where the reactant molecule 'docks in by random collision. - the enzyme is the 'lock' and the initial reactant substrate molecule is the 'key',

- the enzymes active site has a shape closely complementary to the substrate - the substrate locks into the active site, the active site alters its shape holding the substrate more tightly and straining it

- the substrate undergoes a chemical change and a new substance, product, is formed. - the product is released from the active site. - the free unaltered active site is ready to receive a fresh substrate.

The Serine Proteases

(2)

(1)

(3)

(4)

(6)

(5)

(7)

Proposed Enzyme Action of PHB Depolymerase, PhaZBce

Gly
34 N H O O H

His 33
N

CH3

CH3

PHB

O O

PHB
O

:N

N-H

Ser100
228

Asp 175 His

Step 1. As the PHB binds to the substrate binding site of the enzyme, conformational changes in the catalytic triad (Ser-100, His-228, and Asp-175) occur. The aspartate-carboxylate anion forms a strong hydrogen bond with the histidine side chain. This results in the histidine-nitrogen atom with the unshared pair of electrons facing the serine-OH proton. Simultaneously, the hydrogen atom of the serine-OH transfers to the histidine-nitrogen atom (general base) while the oxygen atom forms a bond with the carbonyl carbon of the scissile bond.

Proposed Enzyme Action of PHB Depolymerase, PhaZBce

Gly
34 N H O H

His 33
N

CH3

O H

CH3

PHB

O O

O H

PHB
N

O O

N-H

Ser100 Asp 175

1st tetrahedral intermediate

His

228

Step 2. The first tetrahedral intermediate is formed. In this state, the negatively charged tetrahedral carbon is stabilized by hydrogen bond/s possibly with the main chain NH groups of Histidine-33 and Glycine-34 of the oxyanion hole. The Histidine-228 with its acquired proton (general-acid) can now donate a proton.

Proposed Enzyme Action of PHB Depolymerase

Gly
34

His 33
N H H

CH3

H O O H O

PHB

:N
N-H O

enzyme-acyl intermediate
Ser100
CH3

Asp 175 His

228

PHB

1st product

Step 3. The first product is liberated and the acyl-enzyme intermediate is formed. Hydrolysis of the ester bond of the acyl-enzyme intermediate follows. A water molecule enters the active site. The Histidine-228 (again a general base) abstracts a proton from this water molecule. The water-oxygen atom makes a nucleophilic attack on the ester carbonyl carbon.

Proposed Enzyme Action of PHB Depolymerase, PhaZBce

Gly
34

His 33
N H H

CH3

PHB

O O

H N

+
N-H O

Ser100

2nd tetrahedral intermediate

His
228

Asp 175

Gly
O

34

His 33
N H H

CH3

O O H O

Step 4. The second tetrahedral intermediate is formed. The protonated Histidine-228 (now a general acid) donates back its acquired proton to the tetrahedral intermediate. The second product is liberated. The Serine-100 is restored to its original state for reaction with the next substrate molecule.

PHB

2nd product

:N

N-H

Ser

100

Asp 175 His

228

Classification of Enzymes
Main Class Hydrolases: catalyze the hydrolysis of various bonds Isomerases: catalyze isomerization changes within a single molecule Subclasses Lipases Nucleases Proteases Epimerases Reaction Catalyzed Hydrolysis of an ester group Hydrolysis of a phosphate group Hydrolysis of an amide group Isomerization of a stereocenter

Ligases: join two molecules with Covalent bonding.

Lyases: cleave various bonds by means other than hydrolysis and oxidation Oxidoreductases: Catalyze Oxidation/reduction reactions Transferases: Transfer a functional group

Carbolases Synthetases
Decarboxylases Dehydrases Dehydrogenases Oxidases Reductases Kinases Transaminases

Addition of CO2 Formation of a new bond

Loss of CO2 Loss of H2O Removal of H2O Oxidation Reduction Transfer of a phosphate group Transfer of an amino group

A ribozyme is an RNA molecule that catalyzes either the hydrolysis of phosphodiester bonds in RNAs and the aminotransferase activity of the ribosome

Vitamins and Their Enzyme Functions

Vitamin Water soluble Ascorbic Acid (Vit. C) Thiamin (Vit. B1) Riboflavin (Vit. B2) Pyridoxine (Vit. B6) Niacin Folic acid (Vit M) Vitamin B12 Pantothenic acid Biotin (Vit. H) Fat soluble Vitamin A Vitamin D Vitamin E Vitamin K Enzyme Function Hydrolases Reductases Reductases Transaminases Reductases Methytransferases Isomerases acyltransferases carboxylases Deficiency Symptoms Bleeding gums,bruising Fatigue, depression Cracked lips, scaly lips Anemia, irritability Dermatitis, dementia Megaloblastic anemia Megaloblastic anemia, neurodegeneration Weight loss, irritability Dermatitis, anorexia, depression

Visual system Calcium metabolism antioxidant Blood clotting

Night blindness, dry skin Rickets, osteomalacia Hemolysis of red blood cells Hemorrhage, delayed blood clotting

Recall: Enzymes are biological catalysts. They play a major role in metabolic pathways by lowering the activation energy necessary for chemical reactions to occur.

Recall:
Simple enzymes Conjugated enzymes apoenzyme cofactors such as Fe3+, Mg2+, and Zn2+ coenzymes such as vitamins, NAD+ and FAD+ holoenzymes Substrate Models of enzyme action Lock and Key

Induce-fit
Catalytic site

Factors affecting enzyme activity

1. Temperature
2. pH

3. Substrate concentration

4. presence of inhibitors

enzyme kinetics - the study of the rate at which an enzyme works Why study enzyme kinetics - helps explain how enzyme works - helps predict how enzyme behave in living organisms
-

The Michaelis-Menten equation - one of the simplest and best-known models of enzyme kinetics - named after American biochemist Leonor Michaelis and Canadian physician Maud Menten. - takes the form of an equation describing the rate of enzymatic reactions, by relating initail reaction rate V to [S], the concentration of the substrate S . - equation:

Simple derivation of the Michaelis Menten

Write the rate equation for the formation of product.

V or rate of formation of P = kcat[ES]

Write the rate equation for the formation formation of ES

eq. 1

rate of ES formation = k1[E][S]

Write the rate equation governing the breakdown of ES

eq. 2
eq. 3

rate of ES breakdown = k-1[ES] + kcat[ES]

At equilibrium, rate of ES formation = rate of ES breakdown

k1[E][S] = k-1[ES] + kcat[ES]

Solve the equation in 4 for [ES].

eq. 4
eq. 5

ES = [E][S](k1/(k-1 + kcat)) If KM = (k-1 + kcat)/k1 then, [ES] = [E][S]/KM

eq. 6

.If [ET] = [E] + [ES]

where [ET] = total enzyme concentration [E] = free or unbound form of enzyme [ES] = substrate bound enzyme Then [E] = [ET] - [ES] eq 7 Recall Eq. 6: [ES] = [E][S]/KM

Substituting 7 into 6. [ES] = ([ET] - [ES])[S]/KM [ES]KM = [ET][S] - [ES][S] [ES](KM + [S]) = [ET][S] [ES] = [ET][S]/(KM + [S]) Recall equation 1: V = kcat[ES] Substituting eq. 8 to eq. 1 V = kcat[ET][S]/(KM + [S]) consider Vmax = the maximum rate of forming product = kcat[ET] then V = Vmax[S]/(KM + [S]) Michaelis Menten Equation

eq 8

eq 9

eq 10

The Michaelis Menten Equation

Vi = rate of conversion or initial velocity Vmax = maximum rate of conversion or maximum velocity = the asymptote represents the maximum velocity [S] = substrate concentration (molar) = at low values of [S], the initial velocity,Vi, rises almost linearly with increasing [S]. = as [S] increases, the gains in Vi level off (forming a rectangular hyperbola). Km = Michaelis constant. = substrate concentration at which the rate of onversion is half of Vmax = approximates the affinity of enzyme for the substrate. A low Km indicates high affinity, and a substrate with a low Km will approach Vmax more quickly. = the lower the Km, the lower the concentration of substrate needed to achieve a given rate. = every enzyme has a characteristic Km for a given substrate.

Sample Problems (1) Consider an enzyme with a Michaelis-Menten mechanism, a KM of 0.1 mM and a Vmax of 0.01 mM s-1. At what substrate concentration would the enzyme have an initial velocity of 1 M s-1? Solution: V = (V [S]) / (K + [S])
max M

substituting the given 1 M s-1 = (0.01 mmol s-1 [S]) / (0.1 mM + [S]) solving for [S] 0.001 mM s-1 (0.1 mM + [S]) = (0.01 mM s-1 [S]) 0.0001 mM2 s-1 + 0.001 mM s-1 [S] = 0.01 mM s-1 [S] 0.0001 mM2 s-1 = 0.009 mM s-1 [S] [S] = 0.011 mM (2) At what substrate concentration will an enzyme have an initial velocity of vmax? Solution: V = (Vmax [S]) / (KM + [S])

Substitute V = Vmax, and solve for [S]

Vmax = (Vmax [S]) / (KM + [S]) KM + [S] = (Vmax [S]) / Vmax KM + [S] = 4 [S] [S] = KM / 3

Turnover number (kcat) -maximum number of molecules of subtrate that an enzyme can convert to product per catalytic site per unit of time from Michaelis- Menten equation: Vmax = kcat [E]T Therefore: kcat = Vmax/[E]T kcat = (moles of product/sec)/ (moles of enzyme) = sec-1 Acetylcholinesterase(AChE) = one of the fastest enzymes. = hydrolyzes acetylcholine to choline and an acetate kcat = 7.4 x 105 min1at 25 C, pH = 7.0 Problem 1 The turnover number for an enzyme is 5000 per second How many substrate molecules can be reached by one enzyme molecule in 5 minutes? kcat = Vmax/[E]T 5000/sec = (x/(5min*60 sec/min)/1 molecule x = 1,500,000 molecules

Problem 2 Ten micrograms of enzyme X, which has a molecular weight of 30,000, catalyzes the hydration of 0.30g CO2 in one minute at 37 C under optimal conditions, calculate the turnover number of enzyme X. Solution: kcat = Vmax/[E]T mole CO2 = 0.30g/(44 g/mole) = 0.006818 mole = 6818 mole mole enzyme = 10 g/(30,000 g/mole) = 3.3 x 10-4

Kcat = (6818/min)/3.3 x 10-4 = 2.045 x 107 Problem 3 When 10 micrograms of an enzyme (MW 50,000) is added to a reaction mixture containing its substrate at a concentration 100 times the km, it catalyzes the conversion of 75 micromole of substrate into product in 3 mins. what is the enzyme turn over number (kcat)? Solution:

kcat = Vmax/[E]T

micromole of enzyme = 10 g/(50,000 g/mole) = 0.0002

Kcat = (75 mole/3min)/0.0002 micromole = 125,000/min

Questions:

1. In an enzyme catalyzed reaction, all the enzyme active sites are saturated by substrate molecules at a certain substrate concentration. What happens to the rate of the reaction when the substrate concentration is doubled?
2. Predict the function of each of the following enzymes a. Pyruvate carboxylase b. Alcohol dehydrogenase c. Amino acid reductase d. Cis-trans isomerase e. lactase

3. Describe the effect that each of the following changes would have on the rate of a reaction that involves the substrate sucrose and the enzyme sucrase a. decreasing the sucrose concentration b. Increasing the sucrase concentration c. Increasing the temperature by 200C from its optimum value d. Increasing the pH to 2 pH value lower than the maximum pH

Enzyme Inhibition inhibitor - a substance that interferes with the action of an enzyme and slows down the rate of reaction competitive inhibition - the inhibitor binds to the active site and blocks he substrates access to the enzyme non competitive inhibition - the inhibitor binds to a site other than the active site. The substrate still binds but the enzyme can not catalyze the reaction

other types of inhibition uncompetitive inhibition - the inhibitor binds to the enzyme substrate complex very tightly. the ES complex is not released substrate inhibition - substrate molecules at very high concentrations may crowd the enzyme such that it can no longer function

Michaelis Menten and Inhibitors

Determination of Km and Vmax Linear Transformations of Michaelis Menten Equation 1. Lineweaver-Burk

2. Eadie-Hoffstee

Lineweaver-Burk Plot Eadie-Hoffstee Plot

Problem 1: The data below were observe when cathecol was oxidized by the enzyme o-diphenol oxidase giving rise to dark colored products the concentrations of which were determined in a vis-spectrophotometer. Make the Lineweaver Plot for the reaction. What is the Km and Vmax of the enzyme for the particular substrate?

Trial 1
[S] OD540 = Vi 4.8 mM 0.081 0.21 12

Trial 2
1.2 mM 0.048 0.83 21

Trial 3
0.6 mM 0.035

Trial
0.3 mM 0.020

[1/s]
1/Vi

1.67 29 0.1M
60 50 40

3.33
50

[E]

0.1M

0.1M

0.1M

1/Vmax = 10,so Vmax = 0.10 1/Km = 0.8, so Km = 1.25 mM

1/V

30

20
10 0 -2 -1 0 1 1/S 2 3 4

Lineweaver-Burk Plots for enzymes with inhibitors

Problem 2: The data given below were obtained when the same reaction as in problem 1 was done in the presence of the inhibitor para-phydroxybenzoic acid. What type of inhibitor was PHBA?

Trial 1
[S] OD540 = Vi [1/S] 1/V 4.8 mM 0.06

Trial 2
1.2 mM 0.032

Trial 3
0.6 mM 0.019 1.67 53

Trial
0.3 mM 0.011

0.21
17

0.83
31

3.33
91

[E]

0.1M

0.1M

0.1M
100

0.1M

w/ PHBA
80

1/Vmax = 10, so Vmax remains 0.10.

60

Now, however, 1/Km = 0.4, so Km = 2.50 mM

1/V

40

w/o PHBA

20

Therefore, PHBA is a competetive inhibitor

0 -2 -1 -20 0 1 1/S 2 3 4

Problem 3: The data given below were obtained when the same reaction as in problem 1 was done in the presence of the inhibitor phenylthiourea. What type of inhibitor was phenylthiourea?

Trial 1
[S] OD540 = Vi 1/[S] 4.8 mM 0.04 0.21 25

Trial 2
1.2 mM 0.024 0.83 42

Trial 3
0.6 mM 0.016 1.67 63

Trial
0.3 mM 0.01 3.33 100

1/V [E]

0.1M

0.1M
120 100 80

0.1M

0.1M

1/Vmax = 20, so Vmax = 0.05 1/Km = 0.8, so Km = 1.25 mM

1/V

w/ PTU w/ PHBA

60

w/o inhibitor
40

Therefore PTU is a non competetive inhibitor

20
0 -2 -1 -20 0 1 1/S 2 3 4

Irreversible inhibition -results when a molecule that inactivates enzymes by forming a strong covalent bond to an amino acid side-chain group at the enzymes active site. Thus the enzyme is permanently deactivated Examples of irreversible enzyme inhitors are: nerve gases and organophosphate insecticides

Action:
The human body, contain electrical switching centers called synapses. The body manufactures acetylcholine which turns on the switches The body also produces an enzyme called acetylcholinesterase which breaks down the acetylcholine and turns off the switches. This is how the brain signals information throughout the body, to control respiration, muscle action,and other life functions. A cholinesterase inhibitor, called acetylcholinesterase (ex: nerve gas) binds with the cholinesterase making it unable to breakdown acetylcholine Electrical impulses fire away continuously. Repeated and unchecked firing of electrical signals causes uncontrolled and rapid twitching of muscles, paralyzed breathing, convulsions, and in extreme cases, death.

Why enzyme activity needs to be regulated turning on and turning off 1. Production of large amount of enzyme for a very low amount of substrate is a waste of energy 2. Over production of an enzyme catalyzed material is also a waste of energy Ways of regulating enzyme activity

1. Feedback control using allosteric enzymes 2. Proteolytic enzymes and zymogens

3. Covalent modification Feedback Control -the activation or inhibition of the first reaction is controlled by a product in the reaction sequence

Feedback control is regulated by allosteric enzymes Allosteric enzymes -an enyme with two or more protein chains and two kinds of binding sites (substrate and regulator)

Ex: Feedback Control

Allosteric Enzyme Action

Enzyme Regulation by the Proteolytic Enzymes An enzyme is deactivated or reactivated by removing a portion of the protein A proteolytic enzyme catalyzed the breaking of the peptide bond Zymogens are inactive proteolytic enzymes

Enzyme regulation by covalent modification

- enzyme activity is altered by modifying the structure of the enzyme by covalent attachment Or removal of a chemical group from a particular amino acid of the Protein chain