Enzymes

Compiled by : dr. Santoso

A.Introduction B. Mechanisms of Enzymatic reaction C. Kinetic of enzyme activity D.Factor affecting enzyme activity E. Regulation of enzyme activity

A.Introduction B. Mechanisms of Enzymatic reaction C. Kinetic of enzyme activity D.Factor affecting enzyme activity E. Regulation of enzyme activity

 Most enzymes are Proteins (tertiary and quaternary structures) Act as Catalyst to accelerates a reaction Not permanently changed in the process

What Are Enzymes?

 Are specific for what they will catalyze Are Reusable End in ase

Enzymes

-Sucrase -Lactase -Maltase

A.Introduction B. Mechanisms of Enzymatic reaction C. Kinetic of enzyme activity D.Factor affecting enzyme activity E. Regulation of enzyme activity

How do enzymes Work?

Enzymes work by weakening bonds which lowers activation energy

How do Enzymes Affect Reaction Rates?

Enzymes affect the rates
of reactions by lowering the amount of energy of activation required for the reactions to begin. Therefore processes can occur in living systems at lower temperatures or energy levels than it would require for these same reactions to occur without the enzymes present.

How do Enzymes Bind to Substrates

There are two proposed methods by which enzymes bind to their substrate molecules:

Lock and Key Model Induced-Fit Model

Enzyme-Substrate Complex
The substance (reactant) an enzyme acts on is the substrate
Substrate Joins

Enzyme

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Active Site
A restricted region of an enzyme molecule which binds to the substrate.
Active Site Substrate

Enzyme

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Lock and Key Model

Active site S2
enzyme

S2 S1
SUBSTRATE MOLECULES

enzyme

S1

Products P P
enzyme

ENZYME SUBSTRATE COMPLEX

Enzyme returns from the reaction unchanged and can now react with more substrate.

Induced Fit
A change in the shape of an enzymes active site Induced by the substrate

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Induced Fit
A change in the configuration of an enzymes active site (H+ and ionic bonds are involved). Induced by the substrate.
Active Site substrate Enzyme induced fit
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Induced-Fit Model

Enzyme Cooperativity
Some enzymes have multiple active site. It has been observed that when one substrate molecule binds to a single active site in the inactive form or tense state of the enzyme, a configurational change occurs in the other active sites making them more receptive to other substrate molecules.

A.Introduction B. Mechanisms of Enzymatic reaction C. Kinetic of enzyme activity D.Factor affecting enzyme activity E. Regulation of enzyme activity

Enzyme Kinetics
Expression for enzyme catalyzed reaction:

k1 E+S k-1 ES

k2 E+P

Michaelis-Menten Equation
V0 = Vmax[S] / KM + [S]
Rate increase with [S] Rate levels off as approach Vmax

More S than active sites in E

Adding S has no effect

At V0 = Vmax
[S] = KM

 Vmax occurs when enzyme active sites are saturated with substrate Km (Michaelis-Menten constant) reflects affinity of enzyme for its substrate smaller the Km, the greater the affinity an enzyme has for its substrate

A.Introduction B. Mechanisms of Enzymatic reaction C. Kinetic of enzyme activity D.Factor affecting enzyme activity E. Regulation of enzyme activity

What Affects Enzyme Activity?

Three factors: 1. Environmental Conditions 2. Cofactors and Coenzymes 3. Enzyme Inhibitors

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1. Environmental Conditions
1. Extreme Temperature are the most dangerous high temps may denature (unfold) the enzyme. 2. pH (most like 6 - 8 pH near neutral) 3. Ionic concentration (salt ions)

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Temperature
All enzymes have an optimum temperature at which they work best. If you observe the enzymes activity below the specific temperature it will steadily increase until it reaches the optimum. After the optimum temperature is reached the enzymes activity drops dramatically due to denaturing.

Depending on the species, the range of optimum activity is very broad. Above is a comparison of human enzyme activity with that of bacteria found in hot springs and oceanic vents.

pH
All enzymes have an optimum pH at which they work best. If the pH falls below or rises above the optimum value, enzymatic activity decreases as a result of denaturing.
In the human bodys digestive tract there are variations in pH from area to area. The stomachs juices pH is around 2 (acidic), the enzyme pepsin found in the gastric juices has optimum activity at a pH of 2. The small intestines juices pH is around 8 (basic). The enzyme trypsin found in the small intestines juices has optimum activity at a pH of 8.

Substrate Concentration
The concentration of substrate also has an affect on the rate of enzyme activity. If the concentration of substrate is increased while the concentration of enzyme is constant, the level of enzyme activity will increase until a point of saturation is reached. At this point there are no enzymes available to react with excess substrate and the rate of the reaction stabilizes. No matter if you continue to add substrate, the reaction rate will not increase! Rate of Reaction Point of Saturation, all active sites are filled with substrate.

Increasing Substrate Concentration

2. Cofactors and Coenzymes

Komponen lai yang dibutuhkan enzim supaya dapat berfungsi sebagai katalis disebut kofaktor Inorganic substances (zinc, iron) and vitamins (respectively) are sometimes need for proper enzymatic activity. Example:
Iron must be present in the quaternary structure - hemoglobin in order for it to pick up oxygen.
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Coenzymes are bound at the active site in order to interact with the substrate and play an essential role in the catalysed reaction. They act as carriers of a variety of chemical groups.

Most water-soluble vitamins are components of coenzymes

Vitamin Thiamine (B1) Riboflavin (B2) Coenzyme Deficiency

Thiamine pyrophosphate
FAD+ NAD+ Coenzyme A

Beriberi (weight loss,other problems

Mouth lesions, dermatitis

Nicotinic acid (niacine)

Pantohtinic acid

Pellagra (dermatitis, depression)

Hypertension

Biotin

Biotin

Rash, muscle pain

3. Enzyme Inhibitors

Specific for an enzyme Can be reversible or non-reversible Competitive inhibitors Non-competitive inhibitors

Competitive inhibitors
chemicals that resemble an enzymes normal substrate and compete with it for the active site.
Substrate
Competitive inhibitor
Enzyme

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Noncompetitive Inhibitors
Inhibitors that do not enter the active site, but bind to another part of the enzyme causing the enzyme to change its shape, which in turn alters the active site. Substrate active site altered
Noncompetitive Inhibitor

Enzyme

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Competitive vs. Non-competitive inhibitors

A.Introduction B. Mechanisms of Enzymatic reaction C. Kinetic of enzyme activity D.Factor affecting enzyme activity E. Regulation of enzyme activity

Enzyme activity is regulated by four different mechanisms*

(1) (2) (3) (4) Allosteric control Covalent modification Proteolytic activation Stimulation and inhibition by control proteins

* changes in enzyme levels due to regulation of protein synthesis or degradation are additional, long-term ways to regulate enzyme activity

Allosteric regulation of enzyme activity

Allosteric regulation = the activation or inhibition of an enzymes activity due to binding of an effector molecule at a regulatory site that is distinct from the active site of the enzyme
Allosteric regulators generally act by increasing or decreasing the enzymes affinity for the substrate

Allosteric regulation

Many allosterically controlled enzymse show quaternary structure

Covalent modification regulates the catalytic activity of some enzymes

Can either activate it or inhibit it by altering the conformation of the enzyme or by serving as a functional group in the active site.

Biotin

Biotin serves as a CO2 carrier and is essential for pyruvate carboxylase, participates directly in the catalytic mechanism of the enzyme (as opposed to inducing a conformational change in the enzyme that indirectly affects the activity of the enzyme).

Phosphorylation - an example of regulation by reversible covalent modification of the enzyme

Inserting a negatively charged phosphate group into the appropriate location in an enzyme can induce a conformational change in the enzyme that either increases, or decreases, its activity.

Top 5 reasons why phosphorylation is used to regulate enzyme activity:

1. Phosphorylation is rapidly reversible, making it possible to quickly switch between active and inactive forms of an enzyme. 2. Phosphorylation is relatively inexpensive since it does not require the synthesis of new protein molecules. 3. Results in large Grxn for the phosphorylation reaction. Phosphorylation can shift the conformational equilibrium of a protein by a factor of 104. 4. Phosphorylation/dephosphorylation is rapid and its timing can be adjusted to meet the physiological needs of the cell. 5. Phosphorylation effects can be rapidly amplified via a kinase cascade.

Summary: Covalent modification

1. Covalent modification allows an enzyme to be rapidly activated or inactivated 2. With covalent modification, regulation of a enzyme activity is achieved at low energy costs to the cell (i.e. regulation does not require synthesis of a new enzyme or inhibitory protein). 3. Phosphorylation is a good example of how enzymes are activated and inactivated by covalent post-translational modifications

Proteolytic activation

Proteolytic activation

Such as those involved in protein digestion, blood clotting, and bone and tissue remodeling, must be kept in a completely inactive state until they are needed. These enzymes are synthesized as inactive precursors (known as zymogens or proenzymes) and activated when needed by proteolytic cleavage of a specific peptide bond in the zymogen.

Regulation of digestive enzymes

Digestion of proteins requires simultaneous activation of several digestive enzymes. This is achieved by synthesizing the digestive enzymes as inactive zymogens that are activated by specific proteolysis by trypsin. Trypsin is activated by enteropeptidase catalyzed proteolysis of a unique lysine-isoleucine peptide bond (this is the master switch that turns on the activation of the digestive enzymes).

Pepsinogen is converted to pepsin by autocatalytic proteolysis at pH 2

pepsinogen
(inactive) Secretion into stomach (pH - 2) autocatalytic cleavage of pepsinogen after amino acid 44

Pepsinogen has a low amount of activity at pH 2, allowing it to cleave the peptide bind between amino acids 43 and 44 to generate pepsin, that is much more active than pepsinogen.

pepsin
(active)

Zymogen Pepsinogen Chymotrypsinogen Trypsinogen Procarboxypeptidase Proelastase Prothrombin Fibrinogen Factor VII Factor X Proinsulin Procollagen Procollagenase

Active Enzyme Pepsin Chymotrypsin Trypsin Carboxypeptidase Elastase Thrombin Fibrin Factor VIIa Factor Xa Insulin Collagen Collagenase

Function protein digestion protein digestion protein digestion protein digestion protein digestion blood clot formation blood clot formation blood clot formation blood clot formation plasma glucose homeostasis component of skin and bone remodeling processes during metamorphosis, etc.

Digestive enzymes, blood clotting enzymes, and enzymes involved in bone and tissue remodeling catalyze reactions that would be disastrous if they occurred at inappropriate times or locations. For example, if proteolytic digestion of proteins occurred in the pancreas, they would start digesting the pancreas itself. Similarly, if blood clotting factors are activated when they arent needed, they will initiate blood clotting throughout the body. So, they are synthesized as inactive zymogens and are stored in this inactive state until they are needed.

Blood clot formation - an example of zymogen activations

In tr in s ic P a th w a y
D a m a g e d S u rfa c e K in in o g e n K a llik re in

X II

X IIa

E x tr in s ic P a th w a y
T ra u m a

XI

X Ia

IX

V IIa T is s u e IX a V IIIa fa c to r

V II

Xa Va

P r o th r o m b in

T h r o m b in

F ib r in o g e n

F ib r in X IIIa C r o s s -lin k e d fib r in c lo t

Blood clotting is an excellent example of a proteolytic cascade designed to amplify an external signal (e.g. trauma) and evoke a rapid response (blood clot formation). Thrombin itself is inhibited by antithrombin (a serpin). This provides the body with a mechanism to prevent random blood clot formation beyond the site of injury.

Proteolytic cleavage differs from phosphorylation

1. It can occur outside of cells, since ATP is not needed to convert the zymogen into the active form of the enzyme. 2. It is not a reversible reaction. Inactivation of the active enzyme must occur by either degradation of the enzyme or by inhibition (e.g. due to the binding of an inhibitory protein to the active enzyme).

Stimulation and inhibition by control proteins

Some enzymes have regulatory proteins that bind to them and regulate their activity. cAMP-dependent protein kinase is one examples of this type of regulation.

Serpins - An example of inhibition by control proteins

Once trypsin is activated, we need a mechanism to turn it off when it is no longer needed. Pancreatic trypsin inhibitor is used to shut off trypsin activity

Trypsin (orange) bound to bovine pancreatic trypsin inhibitor (violet). His 57, Asp 102, Gly 193, and Ser 195 in the active site of trypsin are shown in green, red, cyan, and blue, respectively. Lys 15 in BPTI forms a salt bridge with Asp 189 in trypsin in the trypsin:BPTI complex. Binding of bovine pancreatic trypsin inhibitor is essentially irreversible.

pancreatic trypsin inhibitor binds very tightly to trypsin pancreatic trypsin inhibitor is a member of a class of proteins known as serine protease inhibitors (serpins). Serpins are polypeptides that inhibit serine proteases by binding to the active sites of these enzymes.

Elastase is inhibited by 1-antitrypsin

1-antitrypsin inhibits elastase, a serine protease that is responsible for remodeling collagen.

Individuals in which Glu 342 in 1antitrypsin is replaced by a lysine secrete only 15% of the normal levels for 1-antitrypsin, resulting in uncontrolled elastase activity and the breakdown of the alveolar walls in the lung.

Note that although serpins are tight binding inhibitors or serine proteases, they do not form covalent bonds with the serine proteases. In other words, binding of serpins to serine proteases does not involve the formation of covalent bonds between the serpins and the serine proteases.

Summary of regulatory mechanisms

1. Allosteric regulation
ATP activation/CTP inhibition of ATCase sigmoidal kinetics
cAMP activation of cAMP-dependent protein kinase

2. Reversible covalent modification

Phosphorylation
Ser/Thr protein kinases, Tyr kinases, kinase cascades

3. Proteolytic activation
Digestive enzyme, blood clotting factors

4. Protein activators and inhibitors

Serpins

Regulating the rates of enzyme-driven reactions

Cells use inhibitors and activators to turn off and on enzymes
Many enzymes are controlled by an allosteric site remote from the active site

Feedback inhibition Many enzymes are actually regulated by the end products of the reaction they catalyze
Enzyme 1 Enzyme 2 Enzyme 3

Start of pathway

Intermediate

Intermediate

Product

Presence of product inhibits enzyme 1

This prevents too much product from being made

An example of Feedback inhibition

This example demonstrates how an end product can inhibit the first step in its production. Isoleucine binds to the allosteric site of threonine deaminase and prevents threonine from binding to the active site because the shape of the active site is altered. When the level of isoleucine drops in the cells cytoplasm, the isoleucine is removed from the allosteric site on the enzyme, the active site resumes the activated shape and the pathway is cut back on and isoleucine begins to be produced.

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