Enzymes 1 Flashcards
Define and describe enzymes
Enzymes are proteins catalysts, which increase the rate of reactions without being changed in the overall process
- they increase the rate of chemical reactions between substrates (reactants) and products
- can be modified during the reaction, but always return to their original form
They are key players in metabolic pathways
Describe substrate-binding sites as a key concept of enzymes (active sites)
Substrates bind to specific sites through interactions with amino acids, coenzymes and metal ions
- the spatial geometry (3D structure) dictates specificity
Describe activation energy and transition states, in relation to enzymes
The binding of substrates promotes the formation of a transition state which lower activation energy
Describe pH and temperature profiles
Both pH and temperature affect enzyme activity
- Reaction rate increases with temperature
BUT
enzyme stability decreases (denaturation)
Name 2 factors affecting reaction velocity/rate of reactions (in relation to enzymes)
Temperature
pH
Describe how the velocity of reaction can increase with temperature
Reaction velocity increases with temperature
- until a peak velocity is reached
- due to the increased number of substrate molecules having sufficient energy to pass over the energy barrier + form products of the reaction
Describe how the velocity of reaction decreases with higher temperature
Further elevation of the temperature causes a decrease in reaction velocity
- due to temperature-induced denaturation of the enzyme
Describe how pH affects the reaction rate (in relation to enzymes)
The concentration of protons affects amino acids in the enzyme, by altering the interactions between charged R groups in the polypeptide, altering the shape of the enzyme
- leading to denaturation of the enzyme
Describe the enzyme catalytic cycle
3 Basic steps:
- the substrate binds
- product forms
- Product unbinds
Enzymes do not alter the position of the reaction equilibrium, they just accelerate the establishment of this equilibrium
Name the two substrate-binding models
- The ‘Lock and Key’ model (old)
- The ‘induced-fit model (current)
Describe the Lock and Key model of substrate binding
A complimentary 3D surface recognises the substrate
- substrate binds through Hydrogen bonds and hydrophobic/hydrophilic interactions
- Binding can be prevented by steric hindrance and charge repulsions
Describe the ‘induced-fit’ model of substrate binding
As substrates bind, enzymes undergo a conformational change
- side chains of (active sites) amino acids reposition
- binding interactions increase
- not a rigid lock - but a dynamic surface
Name the different energy changes that occur during the reaction
- Activation energy
- Rate of reaction
- Alternate reaction pathway
Define transiton state
The transition state is the point of maximal bond strain
Describe activation energy
( in relation to energy changes during a reaction)
Activation energy
- Difference in free energy between the reactant (substrate) and transition state
- because of high Ea, the rates of uncatalysed chemical reactions are often slow
Describe the rate of reaction (in relation to energy changes during a reaction)
For molecules to react, they must contain sufficient energy to overcome the energy barrier of the transition state
- Without an enzyme, only a small proportion of molecules possess enough energy to achieve the transition state between reactant and product - the rate of reaction is determined by the number of such molecules
So, the lower the Ea, the more molecules have sufficient energy to pass the transition state, so the faster the rate of reaction
Describe the alternative reaction pathway an enzyme provides
An enzyme allows a reaction to proceed rapidly under conditions prevailing in the cell by providing an alternate reaction pathway with a lower Ea
Describe cofactors and coenzymes
Catalytic properties are often dependent on non-peptide molecules called cofactors
- Metal ions and organic coenzymes
- e.g. NAD+, NADP+, Mg2+
Tightly bound cofactors are known as ‘prosthetic groups’
- e.g. FMN, FAD, Fe2+, ZN2+
In humans, coenzymes are usually synthesised from vitamins (so dietary deficiencies can cause problems)
Coenzymes have little to no activity and specificity on their own. Binding to enzymes provides orientation and stabilisation
Describe how a cofactor/coenzyme is involved in activation/transfer
They participate directly in catalysis by forming a covalent bond between the coenzyme’s functional group and the substrate (a separate portion binds to the enzyme)
Describe how cofactors/coenzyme in Oxidation-Reduction reactions
e.g. NAD+ and FAD+
The cofactor is involved in either the oxidation (loss of electrons) or reduction (gain of electrons) of the substrate
Describe how metal ion cofactors help in enzyme activity
Positively charged metal ions act as electrophiles (electron-attracting groups
e. g. Alcohol Dehydrogenase utilises zinc to transfer electrons from ethanol to NAD+
- it forms acetaldehyde and NADH
- Active site contains ‘activated’ serine
- This -ve charge is stabilised by Zn2+
Define isoenzymes
Isoenzymes
- Enzymes that differ in sequences (i.e. different genes) but catalyse the same reaction
- They can have different kinetic parameters (Km and Vmax) and substrate specificities
Describe multi-enzyme complexes
Some enzymes catalysing multiple consecutive steps in metabolic pathways associate to form multi-enzyme complexed
Advantages:
- reduced transit time between steps
- less potential for interference (products acted on by correct enzyme)
Describe diagnostic enzymology
It is the measurement of enzyme activity or concentrations in serum
- can include normal serum enzymes (e.g. clotting factors), secreted enzymes (e.g. pancreatic lipase) and enzymes released by damaged or malignant cells
- Cell leakage can be caused by reduced O2 (ischaemia, impairs energy production), toxic chemicals, microorganisms, immune responses and genetic conditions
Released enzymes are affected by metabolism and excretion, they, therefore, have different serum half-lives:
- intestinal alkaline phosphatase (few hours)
- liver alkaline phosphatase (few days)
Describe the use of diagnostic enzymology in ischaemic heart disease
Heart cells depend on oxygen, which can be restricted by cholesterol-rich atheromatous plaques causing:
- stable angina (angina pactoris)
- myocardial infarction (causing irreversible cardiac cell damage)
Cell contents are released over hours, including creatine kinase (CK) and troponin T or I which can be monitored
Creatine kinase (CK) is a dimer of M (muscle) and B (Brain) subunits
- heart muscle - MM (85%) and MD (15%)
- skeletal muscle - MM (99%)
- Brain, stomach, intestine, bladder mainly BB
As CK-MB is heart-specific, raised serum levels are symptomatic of myocardial infarction
- an increase in total creatine kinase activity can be detected within 6 hours of a heart attack
- CK serum levels peak at 24-36 hours
- CK-MB is detectable within 3-4 hours, with a peak at 100-24 hours
- remains elevated for 2-4 days
Describe enzyme kinetics
E + S [ES] E + P
- Like all chemical reactions, catalysed reactions move towards the equilibrium, where substrates and products are in a steady-state
- The most common approach to analysing enzyme kinetics is using initial rate, v0
- this is affected by [S] and [enzyme], along with external factors (temperature etc.)
Describe how the initial rate is related to Vmax and Km
Plotting v0 against [S] with a fixed [enzyme] yields a hyperbolic curve, which shows how reaction velocity varies with [substrate]
Michaelis-Menten equation
v0 = (Vmax[S])/(Km + [S])
The equation relates initial velocity to [substrate and 2 other parameters]:
- Vmax is the maximum possible rate at that [enzyme]
- Km is the [substrate] resulting in Vmax/2
Define and describe Vmax
It is the theoretical rate of an infinitely high substrate concentration
- where all the enzyme molecules contain a bound substrate
The Vmax is directly proportional to the amount of enzyme used
Describe Km
Km (Michaelis constant)
- Defined as the substrate concentration at which initial rate is 1/2 of Vmax
- enzyme velocity is most sensitive to changes in [Substrate] close to Km
- If Km is known, the [substrate] required to saturate the enzyme can be calculated
High Km = low substrate efficacy
Give assumptions that are being made in order for the Michalis Menten equation to work
- A single substrate reaction where the enzyme-substrate complex (ES) can freely dissociate
- [Substrate]»_space; [Enzyme]
therefore [S]»_space; [ES] - not applicable to all enzymes (e.g. multi-enzyme complexes)
