Russian National Research Medical University
Principles of metabolic control
Enzyme properties and control in cell
homeostasis
Isoenzymes and coenzymes
Medical implications of enzymes
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-andbiological/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/enzym
ekinetics.shtml
Substrate concentration and Enzyme
Activity
http://plantphys.info/plant_physiology/enzymekinetics.shtml
Catalase
• 4 subunit protein
• Decompose hydrogen peroxide
• Contains four porhyrin hem groups with iron
H2O2
H2O + O2
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 classification
Six main divisions (classes) the enzyme
Class
Hydrolase
Isomerase
Type of reaction catalyzed
Hydrolysis
Rearrangement of atoms within
a molecule
Example
Lipase
Phosphoglucoisomerase
Ligase
Joining two or more chemicals
Lyase
Splitting a chemical into
smaller parts without water
Acetyl-CoA
synthetase
Fructose 1,6bisphospate aldolase
Oxidoreductase Transfer of electrons (hydrogen
atoms)
Transferase
Moving a functional group
from one molecule to another
Lactic acid
dehydrogenase
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
Energy
Transition state (X)
Reactants
ΔH
Activation
Energy
Products
www.gcsescience.com
Principles of enzyme catalytic activity.
Reaction energy diagram
X1
X2
Stages of enzymatic reaction
1.
2.
3.
4.
Formation of substrate-enzyme complex (ES)
Catalysis
Formation of product-enzyme complex (EP)
Dissosiation
Formation of substrate-enzyme
complex (ES)
Key-lock interaction (Fisher model)
http://2012books.lardbucket.org/books/introduction-to-chemistrygeneral-organic-and-biological/s21-06-enzyme-action.html
Formation of substrate-enzyme
complex (ES)
Induced fit model
Substrate
Substrate binding changes
conformation of active site
for better catalytic activity
Enzyme
Enzyme-substrate complex
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 catalysis
b. Gain or loss of electrons
c. 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 reactions
b. Transient covalent bonds
c. Charge-charge interactions
• Enzymes can not do:
d. Oxidation -Reduction reactions
e. Carbon group transfers
Holoenzyme: catalytically active enzyme with cofactor.
Apoenzyme: Enzyme without its cofactor
Inorganic cofactors
Inorganic Element
Enzyme
Cu2+
Fe2+; Fe3+
K+
Mg2+
Cytochrome oxidase
Cytochrome oxidase, catalase
Pyruvate kinase
Hexokinase, pyruvate kinase
Mn2+
Mo
Ni2+
Arginase
dinitrogenase
Urease
Se
Zn2+
Glutathione
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 inhibition
Inhibition
Specific
Nonspecific
Irreversible
Competetive
Reversible
Noncompetetive
Uncompetetive
Inhibitor - Any substance that reduces the
velocity of an enzyme-catalyzed reaction
Nonspecific inhibition
1.
2.
3.
4.
5.
6.
pH
Temperature
Heavy metal ions
Ionic strength
Red/Ox chemicals
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 inhibition
Inhibition
Specific
Nonspecific
Irreversible
Competetive
Reversible
Noncompetetive
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
, если
=0
Lineweaver-Burk linearization
Types of inhibition
Specific
Reversible
Competetive
Noncompetetive
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
Succinate
COOH
CH2
COOH
Malonate
HOOC
CH
HC
COOH
Fumarate
succinate dehydrogenase
Competetive inhibition
Substrate
Active Site
Enzyme
Inhibitor
Active Site
Enzyme
Competetive inhibition
Vmax remains the same, but Km is increased
Competetive inhibition
Competetive inhibition
Drug
Enzyme inhibited
Clinical use
Dicoumarol
Vitamin K Epoxide
Reductase
Anticoagulant
Sulphonamide
Pteroid Synthase
Antibiotic
Trimethoprim
Dihydrofolate reductase
Pyrimethamine
Dihydrofolate reductase
Antimalarial
Methotrexate
Dihydrofolate reductase
Anticancer
Lovastin
HMG-CoA-reductase
Cholesterol lowering
drug
Alpha Methyl Dopa
Dopa decarboxylase
Antihypertensive
Neostigmine
Acetyl Cholinesterase
Myastenia Gravis
Antibiotic
HIV protease inhibitors
http://aac.asm.org/content/55/4/1377/F1.expansion.html
Noncompetitive inhibition
Substrate
Enzyme
Active Site
Inhibitor
site
Enzyme
Inhibitor
site
Inhibitor
Active Site
Noncompetitive inhibition
Vmax is decreased, but Km is the same
Vmax
Competitive Inhibitor,
same Vmax, increased KM
Normal Enzyme
Noncompetitive Inhibitor,
same KM, dicreased Vmax
Vi
KM
[S]
Noncompetitive inhibition
Vmax 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 inhibition
a) Reaction
Substrate
Inhibition site
Active site
Enzyme
a) Inhibition
Substrate
Active site
Inhibition site
Inhibitor
Enzyme
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 inhibition
Vmax is decreased, and Km is increased
Vmax
Competitive Inhibitor,
same Vmax, increased KM
Normal Enzyme
Noncompetitive Inhibitor,
same KM, dicreased Vmax
Vi
Uncompetetive Inhibitor,
Km is increased and Vmax is
decreased
KM
[S]
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
Schematic representation of allosteric enzyme activity
http://www.tutorvista.com/content/biology/biology-iii/cellular-macromolecules/enzymesclassification.php
Allosteric Effect
2.0 mM
0.4 mM
Allosteric Effect
http://bcs.whfreeman.com/thelifewire/conten
t/chp06/0602002.html
Feedback inhibition
• The product of a
metabolic
pathway inhibits
is own synthesis
at the beginning
or first committed
step in the
pathway
Carbamoyl phospate
+
Aspartate
ATCase
I
N
H
I
B
I
T
I
O
N
N-carbamoylaspartate
Feedback inhibition
http://highered.mheducation.com/olcweb/cgi/
pluginpop.cgi?it=swf::535::535::/sites/dl/free/
0072437316/120070/bio10.swf::Feedback%20
Inhibition%20of%20Biochemical%20Pathways
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
Isozymes
The enzyme Lactate Dehydrogenase is made of two(B-form and A-Form) different sub units