Principles of metabolic controlEnzyme properties and control in cell

homeostasisIsoenzymes and coenzymes

Medical implications of enzymes

Russian National Research Medical University

Maxim A. Abakumov

Moscow, 2014

Enzymes

• Protein molecule, which 3D structure allows it

to facilitate biochemical reactions that are

hardly to occur under phisiological conditions

• Increase rate of chemical reactions in cell up to

105 – 1015 times

• Active under mild condition under 100 °C,

P =1 atm, pH 7

Enzymes• Contains speciall surface region, called «pocket» critical for

reaction• Pocket is called active site.• Active site shows high specifity to reactants and products.• Catalytic activity can be changed by other biomolecules

(allosteric regulation, inhibitors, activators)

http://2012books.lardbucket.org/books/introduction-to-chemistry-general-organic-and-biological/s21-06-enzyme-action.html

Specific enzyme properties

• pH dependence

• Temperature dependence

• Substrate concentration dependence

pH and Enzyme Activity

Lehninger 2005 Figure 6.17

Temperature and Enzyme Activity

http://plantphys.info/plant_physiology/enzymekinetics.shtml

Substrate concentration and Enzyme Activity

http://plantphys.info/plant_physiology/enzymekinetics.shtml

Catalase

H2O2 H2O + O2

• 4 subunit protein

• Decompose hydrogen peroxide

• Contains four porhyrin hem groups with iron

Inernational enzyme classification

• Consists of 4 numbers separated by periods• 1st number shows to which of the six main

divisions (classes) the enzyme belongs• 2nd number indicates the subclass• 3rd number gives the sub-subclass• 4th number is the serial number of the enzyme

in its sub-subclass

Inernational enzyme classificationSix main divisions (classes) the enzyme

Class Type of reaction catalyzed Example

Hydrolase Hydrolysis Lipase

Isomerase Rearrangement of atoms within a molecule

Phosphogluco-isomerase

Ligase Joining two or more chemicals Acetyl-CoA synthetase

Lyase Splitting a chemical into smaller parts without water

Fructose 1,6-bisphospate aldolase

Oxidoreductase Transfer of electrons (hydrogen atoms)

Lactic acid dehydrogenase

Transferase Moving a functional group from one molecule to another

Hexokinase

Inernational enzyme classification

• Example: glucose oxidase (1.1.3.4)

• 1 – Oxidoreductase (Class 1)

• 1 – CHOH group is oxidised

• 3 – O2 as an electron aceptor

• 4 – Glucose is oxidised

For additional information on enzyme classification: http://enzyme.expasy.org/

Principles of enzyme catalytic activity.Reaction energy diagram

www.gcsescience.com

Transition state (X)

En

ergy Reactants

ActivationEnergy

Products

ΔH

Principles of enzyme catalytic activity.Reaction energy diagram

X1

X2

Stages of enzymatic reaction

1. Formation of substrate-enzyme complex (ES)

2. Catalysis

3. Formation of product-enzyme complex (EP)

4. Dissosiation

Formation of substrate-enzyme complex (ES)

Key-lock interaction (Fisher model)

http://2012books.lardbucket.org/books/introduction-to-chemistry-general-organic-and-biological/s21-06-enzyme-action.html

Formation of substrate-enzyme complex (ES)

Induced fit modelSubstrate

Enzyme Enzyme-substrate complex

Substrate binding changesconformation of active site for better catalytic activity

Types of enzyme catalytic mechanisms

1. Acid-Base Catalysis

2. Covalent Catalysis

3. Metal Ion Catalysis

4. Electrostatic Catalysis

5. Proximity and Orientation Effects

6. Preferential Binding of the Transition State Complex

Acid-base catalysis• Specific Acid-Base Catalysis: Uses only the H+ on

OH- ions present in water. (No other molecules involved) Ions are transferred between water and the intermediate faster than the intermediate breaks down to reactants

• General Acid Catalysis: A process in which partial proton transfer from an acid lowers ΔΔG‡ and accelerates the reaction

• General Base Catalysis: A process in which partial proton extraction by a base lowers ΔΔG‡ and accelerates the reaction

Covalent Catalysis

• Boost reaction through the formation of covalent bonds between substrate and catalyst

• The more stable covalent bond is formed for the transition state the higher is reaction speed

Metal ion catalysis

• Metaloenzymes: contain strongly bound metal ions: Zn2+, Fe2+, Fe3+,Cu2+ and etc

• Metal activated enzymes: temporally actived by metal ions: Na+, K+, Ca2+, Mg2+

Metal ions:a. Bind substrates to orient them for catalysisb. Gain or loss of electronsc. Electrostatically stabilize or shield negative charges

Stabilization of enzyme-transition state complex

Proximity and orientation effects:• Bringing substrate into contact with catalytic groups and

multiple substrates with each other- 5 fold boost• Binding substrates in proper orientation to promote the

reaction – 100 boost

Preferential transition state binding:

• The more tightly an enzyme binds its reaction’s transition state (KT) relative to the substrate (KR) , the greater the rate of the catalyzed reaction (kE) relative to the uncatalyzed reaction (kN) - 107 fold boost

Coenzymes and cofactors

• Some enzymes need any additional nonpeptide components (cofactors) for effective catalysis

• Cofactors can be inorganic or organic• Inorganic: metal ions, iron sulfur clusters• Organic:

1) Tightly bound to enzyme (prosthetic group)

2) Can be released during reaction (coenzymes)

Coenzymes and cofactors• Enzymes can:

a. Carry out acid-base reactionsb. Transient covalent bondsc. Charge-charge interactions

• Enzymes can not do:d. Oxidation -Reduction reactionse. Carbon group transfers

Holoenzyme: catalytically active enzyme with cofactor.Apoenzyme: Enzyme without its cofactor

Inorganic cofactorsInorganic Element Enzyme

Cu2+ Cytochrome oxidase

Fe2+; Fe3+ Cytochrome oxidase, catalase

K+ Pyruvate kinase

Mg2+ Hexokinase, pyruvate kinase

Mn2+ Arginase

Mo dinitrogenase

Ni2+ Urease

Se Glutathione

Zn2+ Carbonic anhydrase, alcohol dehyfrogenase

Organic cofactors

Coenzyme Chemical groups transferred

Dietary precursors

Biotin CO2 Biotin

Coenzyme A Acyl groups Panthothenic acid

5’-Deoxyadenosylcobalamin H, alkyl groups Vitamin B12

Flavin adenine dinuclrotide Electons Riboflavine

Lipoate Electrons, acyl groups

Nicotinamide dinucleotide Hidride ion Nicotinic acid

Pyridoxal phosphate Amino groups Pyridoxine

Tetrhydrofolate One-carbon groups Folate

Thiamine pyrophosphate Aldehydes Thiamine

Enzyme regulation

• Two main mechanisms:

1) Change in enzyme catalytic properties

(inhibition or activation)

2) Change in total amount of enzyme in cell

Types of inhibitionInhibition

Nonspecific Specific

Irreversible Reversible

NoncompetetiveCompetetive Uncompetetive

Inhibitor - Any substance that reduces the velocity of an enzyme-catalyzed reaction

Nonspecific inhibition

1. pH

2. Temperature

3. Heavy metal ions

4. Ionic strength

5. Red/Ox chemicals

6. Organic solvents

• Inhibits all enzyme in same way• Usually goes through denaturation of enzyme and deformation

of active site• Usually occurs under non phisiological conditions

Types of inhibitionInhibition

Nonspecific Specific

Irreversible Reversible

NoncompetetiveCompetetive Uncompetetive

Reversible and irreversible inhibition

• Reversible inhibition – inhibitor can bound

and unbound from enzyme

• Irreversible inhibitor is strongly attached to

enzyme (usually by covalent bondind to active

site)

Irreversible inhibition. Suicide inhibition

• Occurs when inhibitor is transformed in active

site into reactive form

• Aspirin inhibits cyclooxigenase 1 and 2

• Allopurinol inhibits xantine oxidase in the

treatment of gout

• 5-fluorouracil acts as a suicide inhibitor of

thymidylate synthase during the synthesis of

thymine from uridine

Michaelis-Menten equation

Lineweaver-Burk linearization

Lineweaver-Burk linearization

Lineweaver-Burk linearization =0, если

Lineweaver-Burk linearization

Types of inhibition

Specific

Reversible

NoncompetetiveCompetetive Uncompetetive

Competetive inhibition• Inhibitor is usually a substrate-like molecule• Competitive - where the inhibitor competes with the substrate• Can be reduced be increased concentration of substrate.

COOH

CH2

CH2

COOH

succinate dehydrogenase HOOCCH

CHCOOH

COOH

CH2

COOH

succinate dehydrogenase

Succinate

Malonate

Fumarate

Competetive inhibitionSubstrate

Enzyme

Active Site

Inhibitor

Enzyme

Active Site

Competetive inhibitionVmax remains the same, but Km is increased

Competetive inhibition

Competetive inhibitionDrug Enzyme inhibited Clinical use

DicoumarolVitamin K Epoxide Reductase

Anticoagulant

Sulphonamide Pteroid Synthase Antibiotic

Trimethoprim Dihydrofolate reductaseAntibiotic

Pyrimethamine Dihydrofolate reductase Antimalarial

Methotrexate Dihydrofolate reductase Anticancer

Lovastin HMG-CoA-reductaseCholesterol lowering drug

Alpha Methyl Dopa Dopa decarboxylase Antihypertensive

Neostigmine Acetyl Cholinesterase Myastenia Gravis

HIV protease inhibitors

http://aac.asm.org/content/55/4/1377/F1.expansion.html

Noncompetitive inhibition

Substrate

Enzyme

Inhibitorsite

Active Site

EnzymeInhibitorsite

Active Site

Inhibitor

Noncompetitive inhibitionVmax is decreased, but Km is the same

Normal Enzyme

Competitive Inhibitor, same Vmax, increased KM

Vmax

Vi

KM

[ S ]

Noncompetitive Inhibitor, same KM, dicreased Vmax

Noncompetitive inhibitionVmax is decreased, but Km is the same

Noncompetitive inhibition

• Inhibition of cytochrome oxidase by cyanide

• Inhibition of SH-grous by Iodoacetate

• Inhibition of PFK by ATP

Uncompetetive inhibitiona) Reaction

a) Inhibition

Substrate

Substrate

Enzyme

Enzyme

Active site

Active site

Inhibitor

Inhibition site

Inhibition site

Uncompetetive inhibition

• Inhibitor binds only to ES complex• Binding might occur in active site, but prior

substrate bindind is reguired• High concentration of substrate can not

overcome inhibition

Uncompetitive inhibitionVmax is decreased, and Km is increased

Normal Enzyme

Competitive Inhibitor, same Vmax, increased KM

Vmax

Vi

KM

[ S ]

Noncompetitive Inhibitor, same KM, dicreased Vmax

Uncompetetive Inhibitor,Km is increased and Vmax is decreased

Uncompetitive inhibition

Inhibition

Enzyme regulation

• Genetic regulation• Covalent modification• Allosteric regulation• Rate depends on substrate availability • Zymogens, isozymes and modulator proteins

may play a role

Genetic regulation

• Affects on total amount of enzyme in cell• Controlled by gen expression• Hormone controlled • Feedback inhibition or activation• Long term mechanism

Genetic regulation• Increase in enzyme production by increase in gene

expression is called induction.

• Decrease in enzyme production by decrease in gene

expression is called repression

• Induction and repression is used only for gene

expression

• Protein degradation process always occurs in cell

• Total amount of protein is defined by equlibrium

between degradation and synthesid

Genetic regulation. Estrogen receptors

Covalent modification. Controlled proteolysis (zymogens)

• Zymogens (proenzymes) – inactive form of enzyme,

that might be activated

• Zymogens usually are storage form of active enzyme

• Used for rapid regulation of amount of active enzyme

• Usually are activated by proteolysis

Zymogens (proenzymes)

Examples:

• Activation of trypsin and chymotrypsin from

trypsinogen and chymotrypsin by proteolisys

in intestine

• Most proteins in coagulation system are

zymogens

Covalent modification. Phosphorylation/dephosphorylation

• Covalent addition/removal of inorganic phosphate

• Can both increase and decrease reaction speed• Usually catalized by enzyme called kinase

Allosteric regulation

• Key enzymes are regulated by allosteric regulators

• These allosteric effectors usually are located elsewhere in pathway

• Regulators might be activators or inhibitors• Tipicall S-shape (sigmoid) curve• Multisubunit enzymes structure provides

cooperative effect

Allosteric Effect

http://www.tutorvista.com/content/biology/biology-iii/cellular-macromolecules/enzymes-classification.php

Schematic representation of allosteric enzyme activity

Allosteric Effect

0.4 mM

2.0 mM

Allosteric Effect

http://bcs.whfreeman.com/thelifewire/content/chp06/0602002.html

001.swf

Feedback inhibition

• The product of a metabolic pathway inhibits is own synthesis at the beginning or first committed step in the pathway

ATCaseCarbamoyl phospate+

Aspartate

N-carbamoylaspartate

INHIBITION

Feedback inhibition

http://highered.mheducation.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120070/bio10.swf::Feedback%20Inhibition%20of%20Biochemical%20Pathways

002.swf

Glycogen Phosphorylase

• Pi is a positive homotropic effector

• Pyridoxale phosphate is needed as a cofactor• ATP is a feedback inhibitor, and a negative

heterotropic effector (inhibitor)• Glucose-6-P is a negative heterotropic effector

(inhibitor)• AMP is a positive heterotrophic effector (activator)• Activated by phosphorylation• Deactivated by dephosphorylation

Isozymes

• Isozymes – isoforms or different enzymes that catalise

same reaction

• These enzymes usually display different kinetic

parameters (e.g. different KM values), or different

regulatory properties

• Usually they are coded by homologous genes that

have diverged over time

• Provides adaptation of different organs and tissues

Isozymes

Examples:

• Cytochrome P450 family proteins

• Phosphodiesterases

• Hexokinases

IsozymesThe enzyme Lactate Dehydrogenase is made of two(B-form and A-Form) different sub units