Enzyme Kinetics Pt. 2 Flashcards
Michaelis Menten Kinetics Model
- simplest and most well known approach to enzyme kinetics
- equation relating reaction velocity to substrate concentration for a system where the substrate binds reversibly to the enzyme which reacts irreversibly to generate product
Rapid Equilibrium Assumption
- assume ES formation is rapid and achieves equilibrium
- assumes that if Ks is small, the forward reaction is favored, ie. there is strong binding
Steady State Assumption
Assume the concentration of ES complex doesn’t change until all substrate is consumed
The method is based on the assumption that one intermediate in the reaction mechanism is consumed as quickly as it is generated. Its concentration remains the same in a duration of the reaction.
Vmax
The rate of reaction when the enzyme is saturated with substrate is the maximum rate of reaction
Michaelis Menten Graph
- hyperbolic curve
- asymptote gives Vmax
- 1/2 of Vmax gives Km value
- unreliable because of estimation made when finding Vmax values
Km
Km is the concentration of substrate which permits the enzyme to achieve half Vmax
- high Km = low affinity, so more substrate needed to reach Vmax
- low Km = enzyme achieves maximal catalytic efficiency at low substrate concentrations
- k2/k1
Alcohol Consumption
- Alcohol is broken down by acetyl dehydrogenase
- 2 forms of the enzyme: mitochondrial with a low Km and cytosolic with a high Km
- some individuals have a mutated mitochondrial form so only use the cytosolic form
- higher Km = lower affinity for the substrate so less is converted leaving more in the blood - increased alcohol effects
Catalytic Efficiency
Kcat = k2 Vmax = Kcat [Et] Kcat = turnover number, ie. number of reactant processes each active site catalyses per unit time
Kcat/Km
- measure of catalytic efficiency
- gives enzyme substrate preference
- higher number means the enzyme uses the substrate well
Catalytic Perfection
- ratio of Kcat/Km maximal when k2 is much greater than k1 so the product formation is fast compared to its reverse reaction
- therefore, Kcat/Km = k1 and k1 is the rate of ES formation and cannot be faster than the rate of reactant collision
- Kcat/Km only depends on the diffusion controlled limit (10^8-10^9 1/Ms)
- enzyme catalyse a reaction nearly every time a substrate is encountered
LineWeaver Burke Plot
- linearised form of the Michaelis Menten graph
- graph 1/[S] against 1/Vo
- y intercept: 1/Vmax
- x intercept: 1/-Km
- slope: Km/Vmax
LW B Plot Limitations
- most [S] measurements are high values so their reciprocals are crowded on the left hand side of the plot so drawing a straight line is difficult/requires extrapolation
- small [S] values and small errors in Vo gives large 1/Vo errors and hence large errors in kinetic parameter calculations
- 1/V approaches infinity as [S] decreases
- gives more weight to inaccurate measurements made at low concentrations
- gives insufficient weight to more accurate measurements made at high concentrations
Hofstede Eadie Plot
- graph Vo against Vo/[S]
- slope: -Km
- y intercept: Vmax
- x intercept: Vmax/Km
HE Plot Limitations
Can be subject to large error since both coordinates contain dependent variable V but there is less bias on points with low [S]
Hanes Woolf Plot
- graph [S]/Vo against [S]
- slope: 1/Vmax
- y intercept: Km/Vmax
Better fit because of a more even weighting of the data/minimise errors within the data
Reversible Reactions
- MM model assumes the reverse reaction may be neglected
- many reactions are reversible so the product can be converted back to the reactants at a significant rate
Haldens Relationship
- Establish if a reaction will go forwards or backwards
- Keq = [P]eq/[S]eq
Effects of pH
- ph sensitivity of substrate binding
- reduced catalytic efficiency of enzyme
- ionization of substrate
- protein structural changes
Effect of Temperature
- temperature increases the number of molecules with enough energy to reaction
- high temperatures denature proteins
Sequential Bisubstrate Reaction
- all substances combine with enzyme before reaction can occur and products can be released
- single step conversion of substrate to product gives rise to alternate name (single displacement)
- ordered: substrate binding and product release in specific order
- random: substrate binding and product release in any order
Ping Pong Bisubstrate Reaction
one or more products released before all substrate have combined with the enzyme
(double displacement)
Reactions not Obeying MM Kinetics
Allosteric enzymes don’t follow MM kinetics as they have multiple active site
- multiple active sites exhibit property of cooperativity in which the binding of one active site affects the affinity of other enzyme active sites
Inhibitors
Molecules binding to enzymes and decreasing their activity
Can be irreversible or reversible
Competitive Inhibitors
- Competes directly with substrate for active site
- resembles structure of substrate
- non reactive
- lowers concentration of free enzyme available to bind
- Vmax stays the same but Km increases
- Km apparent = Km
Example of Competitive Inhibitor
Sulfanilamide is an antibiotic for kidney infections that mimics p-aminobenzoate, a precursor for folic acid. By preventing folic acid production the bacteria dies. As humans get folic acid through our diet, our cells are unaffected by the antibiotic.
Uncompetitive Inhibitor
- inhibitor binds to proximal site on ES complex only
- can cause active site distortion
- no competition with substrate
- Vmax is affected, so increasing substrate concentration does not achieve Vmax
- Le Chatelier’s Principle opposes ES decrease by making more ES complex so the Km apparent for substrate appear to increase as Km lowers, but the inhibitor doesn’t directly cause this
- Vmax apparent = Vmax/a’
- Km apparent = Km/a’
Example of Uncompetitive Inhibitor
Alkaline phosphatases catalyse release of inorganic P from phosphate esters. Used as diagnostic markers for bone and liver disease.
Mixed Inhibition
- bind at sites that can be proximal or distal from the active site
- inhibitor distorted active site has trouble converting substrate to product before dissociation so apparent substrate binding affinity is lowered
- active site is distorted & enzymatic turnover rate is slowed
- can also bind ES complex
- effective at both low and high [S]
- Vmax apparent = Vmax/a’
- Km apparent = aKm/a’
Noncompetitive
- special type of mixed inhibition where affinity of inhibitor for E and ES is the same
- don’t bind to active site but bind to other part of the enzyme which can be remote from the active site
- extent of inhibition depends entirely on the inhibitor concentration and is not affected by [S]
- Vmax lowered and Km unaffected
Types of Irreversible Inhibitors
- group specific that covalently modify agents
- suicide inhibitors that are mechanism based
- affinity labels
- transition state analogues
Group Specific Covalent Modifying Agents
- react with specific enzyme functional grouns
eg. DIPF: nerve agent that reacts with Ser-OH on acetylcholinesterase in synaptic junctions
Affinity Labels
- structural similarities to substrate guides reagent to active site reaction at active site covalently inactivates enzyme
eg. TPCK has a phenyl group that binds in substrate specificity site at chymotrypsin
Suicide Substrate
- structural similarity to substrate guides reagent to active site and enzyme treats it like a substrate
- chemical mechanism itself leads enzyme to react covalently with inhibitor
- intermediate generated that inactivates the enzyme
- mechanism depends on chemical mechanism of catalysis
eg. Penicillin inhibits transpeptidase enzyme that links cell wall of bacteria
Transition State Analogue
- structurally similar to TS so binding to enzyme is higher affinity than substrate
- must understand catalytic mechanism
- very specific inhibitors
eg. HIV protease is an aspartic protease essential for the virus life cycle. Targeting this using the predicted TS and its analogue inhibits it and makes HIV unable to infect further cells
Enzyme Assay
- used to measure enzymatic activity, study/characterize its kinetics and identify and develop inhibitors
Assay Paramaters
- specificity
- sensitivity
- precision/accuracy
- reproducibility
- setup
Factors affecting an Assay
- salt concentration: can disrupt enzyme bonding and folding
- substance concentrations
- pH dependance: ph should be close to the pka of active site
- activators: cofactors
- temperature dependance
- sample purity
Assay Considerations
- initial rate is being measured
- Vo must be reproducible and dependent on total enzyme concentration
- Need product concentration to be not substantial so we can examine the enzyme affinity for substrate
- control experiments must be done for comparasion
- Vo proportional to [E]: only linear plot is valid as enzyme must be the only limiting factor
Continous Assay
- continuous reading
- multiple measurements
- specific time intervals
- can quickly identify the linear portion of activity
- Vo obtained by slope of linear portion
Spectrophotometric
- continous assay
- suitable if substrate or product absorbs light/UV
- measures absorbance
- very sensitive
eg. absorbance of NADH in conversion of ethanol
Couple Reactions
- continous assay
- used when S or P doesn’t absorb light
- couples reaction to another enzyme that does absorb
- product of 1st reaction is substrate of second
- conversion of PEP to pyruvate is indirectly measured by absorbance of NADH used in conversion of pyruvate to lactate
- reagent precipitation can upset spectrophotometer readings
- must be sensitive to small concentrations
- secondary detection system must not be the RDS
Fluorimetric
- detect fluoresence
- coupled or direct
- very sensitive at low concentrations
- sensitive to light because compound may be unstable
Chemiluminescent
- emission of light from reaction
- extremely sensitive
- usually requires antigen
- hard to quantify and capture all light emitted
Discontinous Assays
- measured concentration in fixed time period
- dilution series for substrate; dilution series for enzyme; dilution series for substrate + enzyme
- reaction must be stopped
- measured with absorbance/radiation/etc
Troubleshooting
- enzyme sequestration: appear as if the enzyme has no activity
- detergent addition masks hydrophobic section to with enzyme binds
- enzyme inactivation
- assay signal interference
