Enzymes Flashcards
What is an enzyme?
- a biological catalyst
- protein
- operate under mild reaction conditions
- specificity
What is a catalyst?
- substance of material which accelerates a chemical reaction without being consumed
Chemical catalysis is in Hydrogenation of ethene on a metal surface
1) Surface chemo-adsorption of H2
2) Surface chemo-adsorption of ethene
3) electrophilic addition
4) rearrangement
5) De-adsorption of ethene product
Equation for chemical catalysis
Kcat / Kuncat
Internal asymmetrical active sites in enzymes
- LYSOZYME
- Reactions with negative ^G occur slowly/not at all due to activation energy
- Energy input needed to convert reactions into unstable molecular forms called Transition State Species
transition state and activation energy determines…
rate of reaction
Effects of heat and pressure in chemical catalysis
Heat - speeds up reactions
Pressure - reduces entropy, increases FOSC
Effects of heat and pressure in Biological catalysis
Heat - increased –> denaturing of proteins
Pressure - increased –> ruptures cells
3 ways enzymes bind to substrates
- lock and key
- induced fit
- transition state stabilisation model
Example of induced fit
hexokinase and glucose
In a transition state diagram what does ^G#uncat equal
Gibbs free energy of activation for the uncatalyzed reaction
Rate constant K depends exponentially on activation energy (transition state diagram)
K = Ae (^G#cat / RT)
- binding to enzyme stabilises transition state, reducing the transition state energy
4 factors that decrease Ae
1) enzyme holds reactants close together (FOSC)
2) enzyme produces microenvironment more suitable
3) enzyme puts strain on existing bonds –> break
4) active site of enzyme directly involved in reaction during transition states (diff pathway)
Stereo specificity
- description of reaction path
- enzymes can be highly specific in binding chiral substrates and catalysing
- stereo-specificity due to enzyme active site geometry
- termed ENANTIO-SELECTIVE
Example of Enantio-selective
- YEAST ALCOHOL DEHYDROGENASE
- ethanol is pro-chiral
- ADH active site determines EtOH binding geometry
- ADH transfers Pro-R hydrogen of EtOH to NAD+
- CH3-CH2OH + NAD+ –> CH3-CH=O + NADH.H+
Geometric specificity
- selective about chemical groups of substrate
- few enzymes are absolutely specific for one substrate
- some work on groups of related molecules e.g. Yeast ADH (primary and secondary alcohols)
- some enzymes are permissive e.g. digestive enzymes (carboxypeptidases)
Coenzymes are…
- metal ions or organic molecules
Cosubstrate is…
- some coenzymes transiently associated with the enzyme
Prosthetic group is…
- cofactors associated with the enzyme known as prosthetic groups
What is a holoenzyme?
catalytically active enzyme-cofactor complex
What is a Apoenzyme?
inactive protein (absence of cofactor)
Precursors and Vitamins
- vitamins that’re precursors are H2O soluble
- Vit A and D (lipid soluble) aren’t components of coenzymes
Enzyme Assays
- measure initial rate of product formation
- of substrate disappearance
Equation for enzyme assay
Rate v = dp/dt or -ds/dt (micromol/min)
Enzyme unit is
- amount which gives 1 micromol prod/min
Katal is
- (SI unit) amount which gives 1mol/sec
Direct Assays
- way of detecting P or S directly dye to property of colour change or spectrophotometer, absorbing at certain wavelengths
- all can be measured continuously
Discontinuous Assays
- reaction stopped at set times P and S measured
- possibly assay to measure P/S would inhibit the enzyme
Example of discontinuous Assay
- glucose measured with Hardings Test
- Reduces CuSO4 to Cu2O when boiled in alkaline
- green colour
- measured in spectrophotometer
Coupled assays
- neither p or s can be measured
- p can be consumed in another reaction and product of that can be measured
- second enzyme must be in excess so that rate limiting step is one being measures
example of coupled assays
alpha - glycerokinase
Michealis Menton Kinetics, equation and assumptions
Equation: V = vmax [s] / Km + [S]
- step ES to EP is irreversible
- [ES] is in steady state
- E>Km : V = Vmax
What is Km?
- Km of enzyme is the substrate conc at which the reaction occurs at half of the maximum rate
- Km is diff between enzymes and for diff substrates
- Km alters with temp and pH
- higher the Km the high [S] needed to reach Vmax
Catalytic Efficiency of enzymes
E + S ES E + P
Vmax = K2 [ET] = Kcat [ET]
- kinetic parameters provide a measure of its catalytic efficiency
- K2 is turnover number Kcat
- numver of reaction processes that each active site catalyses per unit time
Lineweaver-Burk Plot
V0=Vmax [S] / Km + [S]
- linearising it in double reciprocal form
- 1/V0 = Km/Vmax (1/S) + 1/Vmax
- y = mx + c
- slope = Km/Vmax
- Y = 1/vmax
- X = -1/Km
Effects of pH on enzyme activity
- restricted to pH (typically 5-9)
- regulates catalytic efficiency
- substrate ionisation effects
- protein structural changes
Temperate effects on enzyme activity
- increase in temp = increased flexibility in the backbone
- increases activity
- active site effects (reversible)
- denaturation
Types of enzyme regulation
- positive or negative/activation or inhibition
- covalent/non covalent interactions
- covalent binding of regulatory molecule
- non cov interaction of regulatory molecule
- ionic, hydrophobic, van der waals
- can be irreversible/reversible
- post translational mods, protein cleavage, irreversible regulatory molecule binding
- unfold quaternary structure
Non Covalent regulation
- reversible
- highly specific - only target enzyme
- 2 types - simple inhibitors, allosteric regulation
Non covalent regulation binding to…
Enzyme = competitive
Enzyme + Enz Sub = non competitive
Enzyme Sub = uncompetitive
Competitive inhibition
- structure similar to substrate
- occupies active site
- inhibitor + substrate compete
- binding of I + S mutually exclusive
- inhibition reduced by increasing substrate conc
- doesn’t affect vmax of enzyme
- if [S] increases to infinity then all inhibitors displaced
- at all [S], inhibitors will move eq to E from ES
- Km will appear to increase to Kapp
Equation for Competitive inhibition
KI (dissociation constant of EI) = [E] [I] / [EI]
Non competitive inhibition
- Molecule binds at remote site on enzyme in such a way that Kcat is affected
- Km NOT affected
- I + S bind at diff sites
- Vmax reduced as catalytic rate reduced
- point of intercept higher
Equation for non competitive inhibition
V0 = Vmax [S] / ([S] + Km) (1 + [I] / KI)
Uncompetitive inhibition
- only when inhibitor binds with ES complex to make ESI
- cannot yield products
- not reversed by increase in [SUB]
- found in reactions with two or more substrates
- KM decreases
- Increasing [I] diminishes Vmax + Km
- but Km/Vmax remains constant
Allosteric Inhibition
- simple inhibition insufficient, highly sensitive
- requires activation as well as inhibition
- effector needs to be structurally unrelated to S
- effector binds at another site on enzyme
- neg (inhibitor) or positive (activator)
- large amounts of effector needed
- usually non competitive
- effector binding alters protein conformation
- activator improves substrate binding
- inhib reduces this
Allosteric proteins are
- oligomeric
Allosteric Inhibition with regards to V, [S] and [I]
- V against [S] gives sigmoidal curve (doesn’t follow MM kinetics)
- small change in [I] gives big inhibition (no change to Vmax)
- small change in activator conc gives increase in V
Covalent regulation
- even allosteric regulation insufficient
- where activity must be switched off reversibly
- activation or inactivation
- 2 types: - reversible: enzymatically interconvertible form
Irreversible: enzyme activated enzymatically by cleavage
Reversible covalent regulation
- Very sensitive
- Phosphorylation (pyruvate DH) suggested moves enzyme to higher energy state
- adenylation (glutamine synthetase)
Irreversible covalent regulation
- enzyme potentially harmful, these enzymes synthesised as inactive precursors (zymogen)
- e.g trypsin, if immediately active would digest host cells
Oxidoreductases
- oxidation / reduction reactions, transfer H and O atoms or electrons from substrate to another e.g. alcohol DH
Transferases
- transfer functional groups (methyl) from one compound to another e.g. hexokinase
Hydrolases
- catalyse hydrolytic cleavage of C-O, C-N, C-C and phosphoric anhydride bonds. e.g. carboxypeptidase
Lyases
- enzyme cleaving C-C, C-O, C-N, and others by elimination –> double bonds or rings or adding groups to double bonds e.g. pyruvate decarboxylase
Isomerases
- catalyse geometric or structural changes within one molecule e.g. maleate isomerase
Ligases
- catalyse joining of two molecules coupled with hydrolysis of a diphosphate bond e.g. pyruvate carboxylase
Increasing temperature of an enzyme…
- Increases the internal mobility of the enzyme tertiary structure
The steady state assumption in enzyme kinetics assumes that….
[ES] remains constant over the time of measurement
An enzyme catalyses a reaction by…
Decreasing the ΔGǂ of the reaction
