10.11.enzymes
DESCRIPTION
TRANSCRIPT
ENZYMES
ENZYME INHIBITION Inhibitor – substance that binds to an
enzyme and interferes with its activity
Can prevent formation of ES complex or prevent ES breakdown to E + P.
There are 3 categories of enzyme inhibition –
1. Reversible inhibition
2. Irreversible inhibition
3.Allosteric inhibition
REVERSIBLE INHIBITION
Reversible Inhibitors bind through non-covalent interactions (disassociates from enzyme)
E + S <-> ES -> E + PE + I <-> EI
Competitive Uncompetitive Non-competitive
COMPETETIVE INHIBITION Occurs when the inhibitor binds reversibly
to the same site that the S would normally occupy and competes with the S for that site.
Competitive inhibitors are of similar chemical structure to the substrate.
Has effect reversed by increasing substrate concentration
COMPETITIVE INHIBITOR (CI)
•CI binds free enzyme
•Competes with substrate for enzyme binding.
•Raises Km without effecting Vmax
•Can relieve inhibition with more S
COMPETITIVE INHIBITORS CAN BE OVERCOME BY INCREASING [S]
Substrates bind & reaction proceeds
COMPETITIVE INHIBITORS LOOK LIKE SUBSTRATE
NH2C
O
HO NH2S
O
H2N
O
PABA Sulfanilamide
PABA precursor to folic acid in bacteria
Examples: Mehotrexate [anticancer drug] inhibit folate reductase
METHANOL POISONING
Methanol is very toxic and ingestion may lead to optic neuritis, blindness and even death.
Methanol alcohol dehydrogenase Formaldehyde [cause toxicity]
Antidote for methanol poisoning is ethanol [ is a inhibitor of alcohol dehydrogenase]
Ethanol reduces the utilization of methanol and toxicity is averted
Ethylene glycol [antifreeze for automobile] upon ingestion caused CNS depression and Renal damage. Ethanol is used for treatment
UNCOMPETITIVE INHIBITION
An inhibitor which binds only to ES complexes
UI does bind at the active site, but only AFTER the S has bound to the active site so it does not compete with the S.
UNCOMPETITIVE INHIBITOR (UI)
•UI binds ES complex
•Prevents ES from proceeding to E + P or back to E + S.
•Lowers Km & Vmax, but ratio of Km/Vmax remains the same
•Occurs with multisubstrate enzymes
NON-COMPETITIVE INHIBITION
A noncompetitive inhibitor Does not have a structure like substrate Binds to the enzyme but not active site Changes the shape of enzyme and active
site Substrate cannot fit altered active site No reaction occurs Effect is not reversed by adding substrate
NON-COMPETITIVE INHIBITOR (NI)
•NI can bind free E or ES complex
•Lowers Vmax, but Km remains the same
•NI’s don’t bind to S binding site therefore don’t effect Km
•Alters conformation of enzyme to effect catalysis but not substrate binding
NON-COMPETITIVE INHIBITION
Eg.1) Lead poisoning. Pb forms covalent
bonds with SH gps in proteins such as Hemoglobin and causes anemia
2). Poisons like cyanide inhibits cytochrome oxidase in the respiratory chain and causes death
ENZYME INHIBITION IN THE LAB
1/V
1/[S]
no inhibitor
+ inhibitor 1/V
1/[S]
no inhibitor
+ inhibitor
Competitive Non-competitive
Vmax doesn’t change. Km doesn’t change.
1/Vmax
-1/Km
TYPES OF REVERSIBLE ENZYME INHIBITORS
type binding target Km Vmax
Competitive E only =
Noncompetitive E or ES =
Uncompetitive ES only
Summary of inhibition
IRREVERSIBLE INHIBITORS
Irreversible inhibitor binds to enzyme through covalent bonds (binds irreversibly)
IRREVERSIBLE INHIBITORS
H3C O P
O
S C
C
H
O
O CH2CH3
C O CH2CH3
O
S
CH3
CH2
H C
CH3
O
CH3
P
F
O
O C
CH3
H
CH3
Diisopropyl fluorophosphate(nerve gas)
H3C O P
O
S
S
CH3
NO2
parathion
malathion
•Organophosphates•Inhibit serine hydrolases•Acetylcholinesterase inhibitors
IRREVERSIBLE INHIBITION
SUICIDE INHIBITION – Specialized form of irreversible inhibition Original inhibitor (structure analogue) is
converted to more potent form by the same enzyme that ought to be inhibited.
Eg. Allopurinol, an inhibitor of xanthine oxidase, get converted to alloxanthine, a more effective form.
ENZYME SPECIFICITY Enzymes have varying degrees of specificity
for substrates Enzymes may recognize and catalyze:
- a single substrate- a group of similar substrates- a particular type of bond
There are 3 types of enzyme specificity- 1. Stereospecificity 2. Reaction specificity 3. Substrate specificity
1. Stereospecificity
The enzyme can act on only one form of isomers of the substrates.
H
C
H3C COOHOH
H
C
H3C OHCOOH
AB C A
B C
LACTATE DEHYDROGENASE CAN RECOGNIZE ONLY THE L-FORM BUT NOT THE D-FORM LACTATE.
2.REACTION SPECIFICITY Same substrate can undergo different type of
reaction, each catalysed by different enzyme. Amino acid can undergo deamination ,
transamination, etc.
3. SUBSTRATE SPECIFICITY
A. Absolute substrate specificity B. Relative substrate specificity C. Broad specificity
ABSOLUTE SPECIFICITY
Enzymes can recognize only one type of substrate and implement their catalytic functions.
O C
NH2
NH2
+ H2O 2NH3 + CO2
urea
urease
O C
NH
NH2
+ H2O
methyl urea
CH3
Enzymes catalyze one class of substrates or one kind of chemical bond in the same type.
RELATIVE SPECIFICITY
protein kinase Aprotein kinase Cprotein kinase G
To phopharylate the -OH group of serine and threonine in the substrate proteins, leading to the activation of proteins.
OH
OH
HH
OHH
OH
CH2OH
H
CH2OH
HCH2OH
OH H
H OH
O
O
1
1
OH
OH
HH
OHH
OH
CH2
H
CH2OH
HCH2OH
OH H
H OH
O
O
1
1
O
OOH
H
HH
OHH
OH
CH2OH
H 1
sucrose
raffinose
sucrase
BROAD SPECIFICITY
Enzymes acts on closely related substrates E.g. hexokinase acts on glucose,
fructose,mannose
LOCK-AND-KEY MODEL In the lock-and-key model of enzyme action:
- the active site has a rigid shape- only substrates with the matching shape can fit- the substrate is a key that fits the lock of the active site
This is an older model, however, and does not work for all enzymes
INDUCED FIT MODEL In the induced-fit model of enzyme action:
- the active site is flexible, not rigid- the shapes of the enzyme, active site, and substrate adjust to maximumize the fit, which improves catalysis- there is a greater range of substrate specificity
This model is more consistent with a wider range of enzymes
REGULATION OF ENZYME
Many biological processes take place at a specific time; at a specific location and at a specific speed.
The catalytic capacity is the product of the enzyme concentration and their intrinsic catalytic efficiency.
The key step of this process is to regulate either the enzymatic activity or the enzyme quantity.
Maintenance of an ordered state in a timely fashion and without wasting resources
Conservation of energy to consume just enough nutrients
Rapid adjustment in response to environmental changes
REASONS FOR REGULATION
Controlling an enzyme that catalyzes the rate-limiting reaction will regulate the entire metabolic pathway, making the biosystem control more efficient.
Rate limiting reaction is the reaction whose rate set by an enzyme will dictate the whole pathway, namely, the slowest one or the “bottleneck” step.
Zymogen activation
Allosteric regulation
Covalent modification
REGULATION OF ENZYME ACTIVITY
Certain proteins are synthesized and secreted as an inactive precursor of an enzyme, called zymogen.
Selective proteolysis of these precursors leads to conformational changes, and activates these enzymes.
It is the conformational changes that either form an active site of the enzyme or expose the active site to the substrates.
A ZYMOGEN ACTIVATION
Hormones: proinsulin
Digestive proteins: trypsinogen, …
Funtional proteins: factors of blood clotting and clot dissolution
Connective tissue proteins: procollagen
WIDE VARIETIES
A cascade reaction in general
To protect the zymogens from being digested
To exert function in appropriate time and location
Store and transport enzymes
FEATURES OF ZYMOGEN ACTIVATION
Allosteric enzymes are those whose activity can be adjusted by reversible, non-covalent binding of a specific modulator to the regulatory sites, specific sites on the surface of enzymes.
Allosteric enzymes are normally composed of multiple subunits which can be either identical or different.
B ALLOSTERIC REGULATION
The multiple subunits are
catalytic subunits
regulatory subunits
Kinetic plot of v versus [S] is sigmoidal shape.
Demonstrating either positive or negative cooperative effect.
There are two conformational forms, T and R, which are in equilibrium.
Modulators and substrates can bind to the R form only; the inhibitors can bind to the T form.
Properties of allosteric enzymes
[S]
Allosteric enzyme
Allosteric represion
Allosteric activation
ALLOSTERIC CURVE
ACTIVATION OF PROTEIN KINASE
C: catalytic portionsR: regulatory portions
4 cAMP
protein kinase(inactive)
protein kinase(active)
+ +C
C
R
R
C
C
R
R
cAMP
cAMP
cAMP
cAMP
A variety of chemical groups on enzymes could be modified in a reversible and covalent manner.
Such modification can lead to the changes of the enzymatic activity.
C COVALENT MODIFICATION
phosphorylation - dephosphorylation adenylation - deadenylationmethylation - demethylation
uridylation - deuridylationribosylation - deribosylationacetylation - deacetylation
COMMON MODIFICATIONS
Phosphorylation
E-OH E-O-PO3H2
ATP ADP
proteinkinase
phosphorylation
dephosphorylation
H2OPi
Mg2+
phosphatase
Two active forms (high and low)
Covalent modification
Energy needed
Amplification cascade
Some enzymes can be controlled by allosteric and covalent modification.
FEATURES OF COVALENT MODIFICATION
Constitutive enzymes (house-keeping): enzymes whose concentration essentially remains constant over time
Adaptive enzymes: enzymes whose quantity fluctuate as body needs and well-regulated.
Regulation of enzyme quantity is accomplished through the control of the genes expression.
REGULATION OF ENZYME QUANTITY
Inducer: substrates or structurally related compounds that can initiate the enzyme synthesis
Repressor: compounds that can curtail the synthesis of enzymes in an anabolic pathway in response to the excess of an metabolite
Both are cis elements, trans-acting regulatory proteins, and specific DNA sequences located upstream of genes
CONTROLLING THE SYNTHESIS
Enzymes are immortal, and have a wide range of lifetime. LDH4 5-6 days, amylase 3-5 hours.
They degrade once not needed through proteolytic degradation.
The degradation speed can be influenced by the presence of ligands such as substrates, coenzymes, and metal ions, nutrients and hormones.
CONTROLLING THE DEGRADATION
Lysosomic pathway: Under the acidic condition in lysosomes No ATP required Indiscriminative digestion Digesting the invading or long lifetime proteins
Non-lysosomic pathway: Digest the proteins of short lifetime Labeling by ubiquitin followed by hydrolysis ATP needed
DEGRADATION PATHWAY
ENZYMES/PATHWAYS IN CELLULAR ORGANELLES organelle Enzyme/metabolic pathway
Cytoplasm Aminotransferases, peptidases, glycolysis, hexose monophosphate shunt, fatty acids synthesis, purine and pyrimidine catabolism
Mitochondria Fatty acid oxidation, amino acid oxidation, Krebs cycle, urea synthesis, electron transport chain and oxidative phosphorylation
Nucleus Biosynthesis of DNA and RNA
Endoplasmic reticulum
Protein biosynthesis, triacylglycerol and phospholipids synthesis, steroid synthesis and reduction, cytochrome P450, esterase
Lysosomes Lysozyme, phosphatases, phospholipases, proteases, lipases, nucleases
Golgi apparatus Glucose 6-phosphatase, 5’-nucleotidase, glucosyl- and galactosyl-transferase
Peroxisomes Calatase, urate oxidase, D-amino acid oxidase, long chain fatty acid oxidase
UNIT OF ENZYME ACTIVITY
KATAL- mol substrate/sec = katal
INTERNATIONAL UNIT(IU) or STANDARD UNIT(SI unit)
mol substrate transformed/min = unit
1IU = 60 katal
ISOENZYMES
Isoenzymes are different forms of an enzyme that catalyze the same reaction in different tissues in the body- they have slight variations in the amino acid sequences of the subunits of their quaternary structure
For example, lactate dehydrogenase (LDH), which converts lactate to pyruvate, consists of five isoenzymes
ISOENZYMES
ISOENZYMES
EXAMPLES – Isoenzymes of creatine phosphokinase(CPK) or
creatine kinase (CK)it catalyse phosphocreatine to creatineCPK exists in 3 isoenzymes and has 2 subunits M (muscle) , B (brain) or both
CPK1 - BB - brainCPK2 - MB - heartCPK3 - MM - skeletal muscle
APPLICATION OF ENZYMES
A. Enzymes as therapeutic agents – 1. Streptokinase in case of blood clots.
Plasminogen to plasmin in presence of streptokinase then fibrin (clot) to soluble product.
2. Asparaginase in case of leukemias.
B. Enzyme as analytical reagentsuseful in lab.e.g estimation of a plasma glucose by glucose oxidase and peroxidase method
DIAGNOSTIC ENZYMES The levels of diagnostic enzymes in the blood can be used
to determine the amount of damage in specific tissues