Lecture 7 - enzymology 1 Flashcards
what is a catalyst?
a substance that increases the rate of a chemical reaction without itselfundergoingany permanent chemical change
They work by reducing activation energy (ΔG‡) by stabilizing a transition state– they do not affect the reaction equilibrium
They have very high catalytic efficiencies
Exergonic reaction- energy is released e.g. respiration
They are highly specific for substrates and for the nature of the reaction catalysed
Most enzymes work under mild conditions – temperature, pH
Six main classes: grouped by the type of reaction they catalyse (hydrolases, oxidoreductases, lyases, transferases, ligases and isomerases)
how to visualise and measure enzyme activity?
Progress curves allow measurement of
V0: initial reaction rate when [P] is virtually zero
V0: is dependent in enzyme concentration
V0: linearly proportional to [S] if this very low, but independent from [S] if this in excess
Vmax- plateau at equilibrium, not net change in [S] or [P]
Hyperbolic curve: initially linear, reaching a plateau with time
what is the initial rate (V0)?
Initial rate is directly proportional to enzyme concentration v µ [E]
Hence initial rate gives a measure of the amount of enzyme activity, i.e. an assay
Why is ph important for enzyme activity?
Activity is affected by changes in pH because this alters the ionisation state of groups involved in catalysis, pI in relation to pH
Pepsin functions in the stomach (pH ~1.5), trypsin functions in the small intestine (pH ~8).
what is the enzyme active site?
The active site is the location on an enzyme
Where the substrate specifically binds
Where the catalytic reaction occurs
It contains groups that bind the substrates in a defined orientation and groups that help catalyse the reaction
The active site stabilises the transition state
by what models does a substrate bind to the active site?
Lock and key: the active site of the enzyme is complementary in shape to the substrate (classic model)
Induced fit: active site complements the transition state and only fully formed after the substrate is bound to the enzyme
what does the active site look like?
The active site is not a linear sequence of amino acids
Active sites form 3D clefts or crevasses occupying a small part of the enzyme total volume
The active site of lysozyme involves 6 residues from different parts of the polypeptide chain
Tricky to spot from sequence alone
how are enzymes widely used?
household items, biocatalysts, biotech, diagnostics, food and beverages, animal feed, pharmaceuticals, biofuels
what is the effects of temperature on kinetic energy?
Initial rate increases with temperature because a higher proportion of reactant molecules have enough energy to react.
how does temperature have competing effects?
Temperature will affect the stability of enzymes due to their nature as proteins , enzymes are usually more stable in the cold.
Temperature will affect enzymatic activity rate of catalysis
When assayed at high temperatures an enzyme may denature quite rapidly, so the shape of the progress curve will be changed.
The ‘optimum temperature’ for an enzyme is dependent on the nature of the reaction and the type of enzyme.
what is Convergent evolution of enzymes?
Enzymes from evolutionary distant organisms with different overall protein sequence similarities, but with similar mechanisms of action (catalytic triad)
how do enzymes work?
How fast an enzyme catalyses its reaction and what factors affect this
Can provide information about mechanism and about functional role
The Michaelis-Menten equation, Vmax and Km
Measuring Vmax and Km from plots of 1/v against 1/[S] (Lineweaver-Burk plot)
what is the transition state?
Reactions proceed via the transition state. Collisions between reactants only lead to product formation if the reactants have enough thermal energy.
Enzymes work by reducing activation energy, i.e. by stabilising the transition state
what are the experimental measurements of enzyme kinetics?
nad+ does not absorb light at 340 nm
nadh absorbs light at 340 nm
what is the Michaelis-menten equation?
v = vmax (s) / km + (s)
when
v = the reaction rate
vmax = the maximum reaction velocity
km = the Michaelis menten constant
s = substrate concentration
look at the lecture sides for calculations
show the summary of this lecture
Enzymes are biological catalysts
Enzymes reduce the activation energy of the substrates by stabilising the transition state of the enzyme-substrate complex (ES)
Temperature and pH affect and modulate enzymatic function
Enzymatic catalysis takes place at the enzyme active site where the substrate binds with high specificity
Enzyme kinetics can be measured to understand the rate and specificity of reaction (Vmax, Km)
Michaelis-Menten and Lineweaver-Burk plots can be used to study these parameters
Vmax – max reaction rate when E is fully loaded with S
Vmax is directly proportional to [E].
Km – [S] required to give rate of half Vmax
Km is independent of [E]
what is the relationship between Kd and Km
The dissociation constant (Kd) is a thermodynamic parameter and reports the true affinity a ligand (L) has for binding a site on a protein (P).
The Michaelis-Menten constant (Km) is a kinetic parameter, not an equilibrium constant.
It is determined not only by the substrate’s binding affinity, but also by how quickly the enzyme-substrate complex is turned over into product.
For some enzymes catalysis is very slow relative to binding and dissociation, i.e. k2 «_space;k-1, so Km = Kd in these cases. But this is not always true.
what are Km values are relevant to understanding enzymatic function?
Control of glycolysis in liver and muscle are different.
Liver – freely permeable to glucose so cytosolic [glucose] varies around 5 mM according to fed/fasted state, glucokinase Km for glucose is ~ 10 mM.
Muscle – not freely permeable to glucose, cytosolic [glucose] is low, hexokinase Km for glucose is ~ 0.1 mM.
what are the future directions and directed evolution of new enzymes?
How to generate and select enzyme variants with desirable properties, e.g. for uses in the pharmaceutical industry such as to produce derivatives of ‘candidate lead compounds’ with improved properties
how is The hyperbolic dependence of v on [S] is explained by Michaelis-Menten kinetics?
Vmax – max reaction rate when E is fully loaded with S
Vmax is directly proportional to [E].
Km – [S] required to give rate of half Vmax
Km is independent of [E]
what is the effect of increasing (s)
The initial rate of an enzyme-catalysed reaction S → P increases with substrate concentration [S]
In contrast for an uncatalysed reaction the rate increases linearly with [S]
how do you find the rate of reaction?
the rate constant x concentrations of reactants
