- Created by: Jenny Le
- Created on: 06-04-14 23:48
A catalyst enhances the rate of a reaction but is not permanently altered.
They work by decreasing the activation energy (Ea) for a reaction and therefore creating an alternative route which requires less energy.
The structure of the active site of the enzyme is used to optimally orient the subtrate for reaction (amino acid R group position).
In order for molecules to interact, they must collide with the correct orientation and activation energy.
Reactions start off quick to produce product and rapidly decline as the number of free molecules decrease.
- LOCK AND KEY MODEL - the enzyme is assumed to be the lock and the substrate the key, the two are made to fit exactly. However this model fails to account for the fact that proteins change their conformations to accommodate a substrate molecule.
- INDUCED FIT MODEL - the enzyme conformation changes to accommodate the substrate molecule, the enzyme and substrate do not fit exactly until the collision and therefore reaction takes place.
- KINETICS - the field of chemistry that studies the rate and mechanism of a reaction
- RATE - usually measured in terms of how many moles of reactant or product are changed per time period.
- MECHANISM - a detailed step-by-step description of how a reaction occurs at the molecular level.
Enzyme activity units
RATES are measured in umol/min etc.
International Unit of Activity, IU = 1 umol/min
The SI unit is the KATAL (Kat) = 1 mol/sec
Equal to: nKat = 1 nmol/sec
Km - Michaelis Constant
Km is an important kinetic constant for an enzyme/substrate reactioin and is a measure of AFFINITY and hence EFFICIENCY.
The LOWER the Km, the GREATER the affinity of the enzyme for the substrate and the more efficient it is.
Measuring enzyme activity
- Enzymes are measured by determining their activity in an enzyme ASSAY
- Measuring the rate of disappearance of substrate or appearance of product.
- The reaction rate slows down as the substrate disappears - SUBSTRATE DEPLETION
- The initial rate of the reaction (velocity, Vo) is proportional to the enzyme concentration
Inhibitors interfere with enzyme action. They can be reversible or irreversible. The three kinds of reversible inhibitors are:
A competitive inhibitor has a similar structure to the substrate and binds to the active site of the enzyme.
A non-competitve inhibitor does not look like the substrate and binds to the enzyme away from the active site.
An un-competitive inhibitor binds only to the enzyme-substrate complex.
A competitive inhibitor competes with the substrate for the active site, its influence can be overecome with large concentrates of substrate. therefore the Vmax remains constant but the reaction is slower.
Since the velocity is slower compared to normal substrate concentrations, the Km increases.
The concentration of free enzyme is reduced.
A non-competitive inhibitor will bind to the enzyme away from the active site and results in a conformational change to the enzyme.
The substrate can still bind to the enzyme but cannot catalyse the reaction therefore, no product is formed.
The velocity of the reaction is slowed down at all substrate concentrations.
Therefore the Vmax is permanently lowered but the Km values stays constant.
An un-competitive inhibitor binds to the enzyme-substrate complex after ES binding.
The formation of the inhibitor's binding site only forms when the enzyme and substrate have interacted.
The enzyme-substrate-inhibitor complex does not produce any products.
Both Vmax and Km values decrease as [inhibitor] increases.
Irreversible enzyme inhibition
Irreversible inhibitors form stable covalent bonds with the enzyme, e.g. alkylation or acylation of an active site side chain.
There are many naturally occuring and synthetic irreversible inhibitors, e.g. poisons; cyanide, lead.
These inhibitors can be used to identify the amino acid residues at enzyme active sites.
Incubation of these inhibitors with enzyme results in loss of activity
Uric acid is produced by an enzyme called xanthine oxidase which oxidases the reaction:
HYPOXANTHINE (FROM NUCLEIC ACIDS) ---------> URIC ACID
In some cases, this enzyme becomes overactive and they produce too much uric acid.
Uric acid is not very soluble and so high concentrations of it in the blood can lead to a precipitate depositing in the capillaries of feet which is a condition known as gout. It can also precipitate in the urine leading to kidney stones.
A drug known as ALLOPURINOL (competitive inhibitor) prevents the binding of hypoxanthine to the enzyme xanthine oxidase and therefore the product of uric acid can no longer be produced as the reaction cannot be catalysed.
Inhbitiors in pharmacology
A statin known as Zocor (simvastin) is a popular competitive inhibitor which inhibitorsn an ezyme in the pathway of cholesterol biosynthesis.
It is similar to the substrate, HMG-CoA and lowers blood cholesterol in people that produce too much of this lipid.
Many anti-HIV drugs are competitive inhibitors of the enzymes which polymerise the viral nucleic acid.
Competitive inhibitors are very important in pharmacology. Biochemists are also able to use analogs of true substrates in order to discover the nature of the enzyme's active site to determine which groups on the substrate are being recognised by the active site.
Methods of Enzyme Regulation
NON-COVALENT ALLOSTERIC REGULATION
Allosteric enzymes have a second regulatory site (allosteric site) distinct from the active site.
Allosteric inhibitors or activators bind to this site and regulate enzyme activity via conformational changes.
Allosteric enzymes have quaternary structure and are at important crossroads in metabolism.
Allosteric effectors bind to multisubunit enzymes such as aspartate transcarbamoylase thereby inducing comformation changes that alter the enzyme's activity.
ATCase catalyses the reaction between aspartate and carbomolyl phosphate. The reaction leads to the synthesis of nucleobases needed for DNA and RNA synthesis.
Velocity as a function of [aspartate] gives a sigmoidal plot.
Role of co-operation of binding in regulation
Addiiton of modulator alters enzyme activity
Activators can lower Km and inhibitors can raise Km.
Activators and inhibitors are called modulators or effectors.
Allosteric ligands (effectors) can be positive or negative
CTP is an INHIBITOR of ATCase activity or a negative effector (pyrimidine)
ATP is an ACTIVATOR of ATCase activity or a positive effector (purine)
Phosphofructokinase-1 (PFK-1) is an allosteric enzyme which catalyses an early step in glycolysis.
Phosphoenol pyruvate (PEP) is an intermediate near the end of the pathway and also is an allosteric inhibitor for PFK-1
ADP is an allosteric activator of PFK-1
- ADP activates PFK-1 in order for glycolysis to take place.
- PEP inhibits PFK-1 for regulatory control
High [PEP] inhibiting PFK-1 = Low [ADP] = REDUCED GLYCOLYSIS
Low [PEP] = High [ADP] activating PFK-1 = INCREASED GLYCOLYSIS
ADP lowers the Km without affecting Vmax, for a given F6P concentration, the Vo is larger in the presence of ADP.
PEP raises the Km without changing Vmax