enzyme i learn 24 april 2014

24th April 2014

Enzyme

Introduction

Highly selective protein catalyst greatly accelerating both the rate and specificity of metabolic reactions

Increase the velocity of chemical reaction w/out being changed

Abbreviations: E = enzyme; S = substrate; P = product; I = inhibitor

The below topics will be covered in this lecture:

1. E- nomenclature

2. Properties of Es

3. How Es work

4. Factors affecting reaction velocity

5. Michaelis Menten equation

6. Inhibition of E activity

7. Regulation of E activity

Category: Es by function

Enzyme Commission number (EC number), a numerical

classification scheme for Es, based on the chemical reactions

they catalyze;

1. EC 1 Oxidoreductases: oxidation/reduction reactions

2. EC 2 Transferases: transfer a C-, N- or P- containing functional gp

3. EC 3 Hydrolases: hydrolysis of various bonds; addition of H2O

4. EC 4 Lyases: cleave C-C, C-S and certain C-N bonds by means other than

hydrolysis and oxidation

5. EC 5 Isomerases: isomerization changes within a single molecule

6. EC 6 Ligases: join two molecules with covalent bonds; catalyze formation

of bonds between carbon and O,S, N coupled to hydrolysis of high energy

phosphates

Hydrolase

Hydrolases (EC 3) can be further classified into several subclasses,

based upon the bonds they act upon:

EC 3.1: ester bonds (esterases: nucleases, phosphodiesterases,

lipase, phosphatase) eg: alkaline phosphatase Practical 4

EC 3.2 : sugars (DNA glycosylases, glycoside hydrolase)

EC 3.3 : ether bonds

EC 3.4: peptide bonds (Proteases/peptidases)

EC 3.5 : carbon-nitrogen bonds, other than peptide bonds

Etc..

http://en.wikipedia.org/wiki/Hydrolase

Format of number

Every E code consists of the letters "EC" followed by four numbers separated by periods. Those numbers represent a progressively finer

classification of the enzyme.

Eg: tripeptide aminopeptidases; EC 3.4.11.4

Reaction catalysed: release of the N-terminal residue from a tripeptide;

Require cofactors Zinc

EC 3 - Es are hydrolases use H2O to break up some other molecule

EC 3.4 - are those hydrolases that act on peptide bonds

EC 3.4.11 - cleave off the amino-terminal AA from a polypeptide

EC 3.4.11.4 - cleave off the amino-terminal end from a tripepeptide

http://enzyme.expasy.org/EC/3.4.11.4

http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.4.11.4 - BRENDA Enzyme Database

Properties of enzymes active site

1. Active site is the small portion of E where S molecules bind

and undergo a chemical reaction

Properties of enzymes specificity

2. Specificity due to:

Complementary shape, charge & hydrophilic/hydrophobic

characteristics of Es and Ss

Properties of enzyme catalytic efficiency

Energy changes during the chemical reactions with and without E

http://en.wikipedia.org/wiki/Enzyme

Reaction coordinates are often plotted against free energy to demonstrate in

some schematic form the potential energy profile associated to the reaction.

Energy barrier separating the reactants and the products

Thermodynamics

Properties of enzymes catalytic efficiency

Ss need a lot of potential energy to reach a transition state (highest potential energy along this reaction coordinate) which then decays into Ps.

The E stabilizes the transition state, reducing the energy needed to form Ps.

Like all catalysts, Es work by lowering the activation energy for a reaction

Thus, dramatically increasing the rate of the reaction; Ps are formed faster and reactions reach their equilibrium state more rapidly.

Transition state

The transition state of a chemical reaction is a particular

configuration along the reaction coordinate.

It is defined as the state corresponding to the highest potential

energy along this reaction coordinate.

At this point, assuming a perfectly irreversible reaction, colliding

reactant molecules will always go on to form products.

It is often marked with the double dagger symbol.

Eg: the transition state occurs during the SN2 reaction of

bromoethane with a hydroxyl anion.

Properties of enzymes holoenzymes

3. Some Es require non-protein molecules (helper) for enzymatic activity

Non-protein components:

1. Cofactor inorganic-metal ion; Zn2+, Mg2+, Fe2+

2. Coenzyme organic molecules

a) Cosubstrates dissociate from E in an altered state (eg: NADH, NADPH, ATP)

b) prosthetic group permanently associated with the E and returned to its original form; FADH)

Holoenzyme active E with its non-protein components

Apoenzyme inactive E without its non-protein components

Properties of enzymes location within cell

4. Location within cell

- Compartmentalization serves to organizes the thousands of Es present in the cell into purposeful pathways

- isolate the reaction, S or P from other competing reactions

- favorable environment for the reaction;

- Enzymes in urea cycle

cytosol (arginase exclusive in liver cell) and

mitochondria (ornithine carbamoylase)

Properties of enzymes regulation

5. Regulation

- Es activity can be regulated (slide 35)

1. Induction and repression of E synthesis

2. Regulation of Es by covalent modification

3. Regulation of allosteric Es

Factors affecting reaction velocity - definition

1. Reaction velocity (v)

the number of S molecules converted to P per unit time; usually expressed

as mol of P formed per minutes

(molmin-1)

2. Enzyme activity

amount of P produced per unit time (minutes) per mg of enzyme

(molmin-1 mg-1)

Factors affecting reaction velocity

1. Temperature

2. pH

3. [S]

4. Salt concentration

a) Activation energy of S

b) Denaturation of E

c) Chemical interaction between AA residue at

the active site of E and chemical gp of S

Effects of Temperature

Generally temp. V0

Number of molecules having sufficient energy to pass over the energy barrier and form the Ps of the reaction

Further temp. lead to a sharp decrease in reaction velocity as a result of temp-induced denaturation of the E active site.

The "optimum" temp for human Es is between 35 and 40C; average temp. is 37C; Es start to denature at 40C.

Es from thermophilic archae found in the hot springs are stable up to 100C.

Effects of pH

Most Es are sensitive to pH and have specific ranges of activity; all E have an optimum pH

pH effect on the ionization of the active site; E and S have specific chemical groups in either an ionized or un-ionized state in order to interact.

pH can lead to denaturation on E, altering the three dimensional shape of the E by breaking ionic, and hydrogen bonds

The pH at which maximal E activity is achieved is different for different Es and often reflect the [H+] at which the E functions in the body.

Eg: pepsin, a digestive E in the stomach is maximally active at pH 2

Effect of [S]

Increasing the [S], increases the rate of reaction velocity

However, E saturation seen after reaching Vmax

The leveling off of the reaction rate at high [S] reflects the saturation with S of all available binding sites on the E molecules present

The graph of the reaction rate will plateau

Effect of salt concentration

Most Es cannot tolerate extremely high salt concentrations.

The ions interfere with the weak ionic bonds of proteins.

Typical Es are active in salt concentrations of 1-500 mM;

exceptions for the halophilic algae and bacteria.

MichaelisMenten kinetics

Es kinetics is the investigation of how Es bind Ss and turn them into Ps

MichaelisMenten kinetics is one of the simplest and best-known

models of E kinetics

Es reactions occur in two stages;

1. S binds reversibly to the E, forming the ES complex.

2. The E then catalyzes the chemical step in the reaction and releases

the P, regenerating free E

Michaelis-Menten equation

The model takes the form of an

equation describing the rate

of enzymatic reactions, by relating

reaction velocity (v) to the [S].

Reaction velocity

The number of S molecules converted

to P per unit time

http://en.wikipedia.org/wiki/Michaelis-

Menten_kinetics


The model takes the form of an

equation describing the rate

of enzymatic reactions, by relating

reaction velocity (v) to the [S].

Vmax = maximum rate achieved by the

system, at maximum (saturating) [S]

Km is the [S] at which the reaction rate

is half of Vmax


With respect to [S], the rate of reaction

(within the specified [E]) is said to be:

- First order - when the reaction

velocity is approximately proportional

to the [S]; occur when [S] >Km


Km is an inverse measure of the

substrate's affinity for the E;

-Small Km indicates high affinity

of the E for the S, because a low [S]

is needed to half saturate the E, that

is, to reach a velocity that is Vmax

Km value does not vary with the

[E]

Practical 4 - Enzyme

Alkaline phosphatase (ALP, ALKP) (EC 3.1.3.1) is a hydrolase E responsible for removing phosphate groups (p-nitrophenylphosphate) from many types of molecules, including nucleotides, proteins and alkaloids.

The process of removing the phosphate group is called dephosphorylation.

AP are most effective in an alkaline environment

Practical 4-Enzyme-hyperlink.doc

Practical 4 - Enzyme

Effect of pH

Effect of temperature

Calculate the enzyme activity (molmin-1 mg-1)

Practical 4 - Enzyme - Questions

1. Draw a calibration graph for absorbance versus p-nitrophenol produced by the enzymatic reaction.

2. Use the graph to determine the amount of p-nitrophenol produced by the enzymatic reaction.

3. Calculate the enzyme activity (molmin-1 mg-1).

4. Draw a graph of enzyme activity versus pH.

5. Draw a graph of enzyme activity (V) versus temperature.

6. Discuss the results obtained.

7. What happens to the enzyme activity during fever?

8. Give 2 examples of active enzymes in the gut and duodenum. Explain factors that influence the enzyme activity.

Calibration graph for absorbance versus p-nitrophenol

Calculate the enzyme activity (mol min-1 mg-1)

(Absorbance reading for each reaction is converted to mol unit- from graph

extrapolation)

Effect of pH Calculate the enzyme activity (mol min-1 mg-1)

Effect of pH

Do the same for temperature parameter

Effect of temperature

Calculate the enzyme activity (mol min-1 mg-1)

Inhibition of E activity

E inhibitor is a molecule which binds to Es and decreases their activity

Inhibitor binding is either reversible or irreversible

Irreversible inhibitors usually react with the E and change it chemically (e.g. via covalent bond formation); modify key AA residues needed for enzymatic activity

Reversible inhibitors bind non-covalently to the E, E-S complex, or both. Comprise of competitive inhibition and noncompetitive inhibition

Effect on the value of Vm and Km ?

http://en.wikipedia.org/wiki/Enzyme_inhibitor

Competitive inhibition

S and I compete for access to the E's active site

S and I cannot bind to the E at the same time

Inhibition can be overcome by sufficiently high [S]

Vmax remain constant

Apparent Km increase as it takes a higher [S] to reach the Km point, or

Vmax

Noncompetitive inhibition

I and S bind at different sites on the E

I bind either free E or ES complex

Binding of the I to the E reduces its activity but does not affect the

binding of S.

Vmax decrease due to the inability for the reaction to proceed as efficiently

Km remain constant as the I do not interfere with the binding of S to E.

Regulation of E activity

1. Induction and repression of E synthesis

2. Regulation of Es by covalent modification

3. Regulation of allosteric Es


1. Cells can regulate the amount of E present by altering

the rate of :

E synthesis: induction and repression of E synthesis; often those that are needed at only one stage of development or under selected physiologic

condition (eg: high blood glucose level elevated level of insulin increase in the synthesis of key E involved in glucose metabolism)

E degradation: refer lecture notes: AAs and Protein 2 dated 3rd April

2014; protein turnover rates; major E systems for regulating the

concentration of particular proteins; chemical signals for protein

degradation

* Leads to alteration in the total population of active sites


2. Covalent modification

Most frequent one of the primary ways in which cellular processes is regulated is protein (E) phosphorylation

Involved the addition or removal of phosphate gps from specific AA residues of the E: serine, threonine or tyrosine residues

Phosphorylation of glycogen phosphorylase (degrades glycogen) increases activity

Phosphorylation of glycogen synthase (synthesizes glycogen) decreases activity


3. Allosteric regulation (activation or inhibition)

-Refer to the regulation of an E or other protein by noncovalent binding of an effector molecules at the protein's allosteric site (that is, a site other than the protein's active site).

-Effectors that enhance the protein's activity are referred to as allosteric activators, whereas those that decrease the protein's activity are called allosteric inhibitors.

http://en.wikipedia.org/wiki

/Allosteric_enzyme


Homotropic effectors when the S itself serves as an effector

Presence of a S molecule at one site on the E increases/decreases the catalytic properties of the other S binding sites; their binding sites exhibit positive/negative cooperativity

A macromolecule exhibit cooperative binding if its affinity for its ligand changes with the amount of ligand already bound

Km?, Vmax ?


Cooperative binding requires that the

macromolecule have multiple binding

sites for substrate and regulatory

molecules, since cooperativity results

from the interactions between binding

sites

Exhibit sigmoidal curve when V0 is

plotted against [S]


Presence of an allosteric effector can

alter Km (affinity of the E for its S) and

Vmax (maximal catalytic activity of the E)

or both

Fig: Effects of negative or positive +

effectors on an allosteric enzyme.

A. Vmax is altered.

B. The substrate concentration that gives half-maximal velocity (K0.5) is altered.


Heterotrophic effectors when

the non S serves as an effector

Fig: Feedback inhibition of a metabolic pathway

The E that convert DE has an allosteric site that bind G (the end product)

Allosteric enzymes frequently

catalyze the committed

early step in a pathway

Sum

mar

y

End of lecture

Thank you

Habit 1: Be Proactive Habit 2: Begin with the End in Mind

Habit 3: Put First Things First

Habit 4: Think Win/Win

Habit 5: Seek First to Understand, Then to Be Understood

Habit 6: Synergize

Habit 7: Sharpen the Saw

Stephen R. Covey, The 7 Habits of Highly Effective People

enzyme i learn 24 april 2014

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