2.3 Enzyme kinetics Flashcards
Explain enzyme thermodynamics vs enzyme kinetics
THERMODYNAMICS : ΔG governs how if the reaction is thermodynamically favourable (ΔG < 0) - investigates reaction E changes (feasible / unfeasible)
KINETICS : Ea governs if reaction procedes (Ea <) - investigates reaction rates (fast / slow)
What characteristics of a reaction needed to be able to investigate reaction kinetics?
Some kind of change that can be measured quantitatively:
- pH
- colour change
- mass change
- precipitate
The basic reaction rate equation, what are the units
conc. / t
ex: mol dm-3 s-1; mg ml-1 min-1; molecules ml-1 min-1; Torr s-1
How to figure out the rate at a specific time using the rate graph?
Rate at specific time - tangent at specific point - differential equation
What is the relationship of reaction rate and stoichiometric equations?
O2 + 2H2 -> 2H2O
- Rate can be calculated from stoichiometric equations - ratios in which reactants / products are lost / produced
- Rate law CANNOT be calculated from stoichiometric equatinos!!
What is the equation for rate determination from stoichiometric coefficient va?
aA + bB -> cC + dD
- rate of conc [A] against time t
- va : < 0 if reactants (add -) ; > 0 if products
How are rate laws determined?
Rate laws for a specific reaction are only determined EXPERIMENTALLY
What is the general rate law equation? Explain components
- x is order of reaction with respect to A
- y is order of reaction with respect to B
- x+y is order of the whole reaction
- k is rate constant (independent of conc. - specific for particular reaction) - k units depend on order
What are the possible methods to solve >3 order equations?
When rate laws become >3 -> difficult to integrate:
- isolation method
- differentiation method
Explain differential method to calculate reaction orders
- used when orders are >3: for enzyme reactions by changing substrate conc
How does change in temperature affect reaction kinetics?
- T affects collision frequencies - one of requirements for reaction to occur
- increase in T widens the distribution of Maxwell-Boltzmann energy curve - more molecules have enough E to overcome Ea and react
Explain isolation method to calculate reaction orders
- used when orders are >3: can’t integrate because don’t know if one variable conc is constant - set one constant by using excess amounf of it => pseudo second order rate
- keff - ??
Can rate laws be predicted by stoichiometric equations?
NO, rate laws are determined only experimentally
Explain what is Arrhenius equation used for?
Arrhenius equation describes the effect of temperature on rate constant (k) and reaction rate
What are Arrhenius parameters?
- A (pre-exponential factor)
- Ea
Which graph is used to determine reaction Ea depending on T?
What is a steady state approximation?
Approximation that intermediate product is constant in a two step reaction - used up as soon as made - allows to figure out the rate of the reaction only using reaction rates (k1, k2) and reactants conc ([A], [B])
Full explanation: BioChem2 Enzyme energetics and kinetics 4 notes
What change in a reaction allows enzymes to act as catalysts? What are the mechanisms for the change?
Lowering activation energy:
- by binding a transition state - stabilising intermediate
- by changing the reaction pathway
Which equation allows to account enzyme function: reaction rate change linking yo activation energy change
Arrhenius equation
What graph is used ot depict enzyme catalysed reaction:
S + E -> ES -> E+P
What does high/low Km represent?
High Km - weak E-S binding: need more [S] to occupy half active sites
Low Km - strong E-S binding: need less [S] to occupy half active sites
What is Kcat?
Kcat - turnover number (# of S molecules converted into P by 1 molecule of E active site per unit time when E is fully saturated with S) - rate of product formation ES -> E+P (second reaction - equivalent to k2)
Kcat = Vmax / [E]t
How can enzyme efficiency be evaluated?
Enzyme efficiency = Kcat / Km
What is Lineweaver-Burke plot? Why is it useful?
Lineweaver-Burke plot (linear equation) can be used to solve Michaelis-Menten equation
What is the requirement for SSA application?
At the start of the reaction [S] must be high and** [P] low**
How enzyme kinetics is determined in laboratory?
(1st practical: enzyme activity measured with spectrophotometer)
Initial rates method:
- measure V from the start of reaction - V will change with time until dynamic equilibrium is reached (rate forward = rate reverse reaction) - d[P]/dt=0 - no change
How coupled enzyme kinetics is determined in laboratory?
(1st practical: enzyme activity measured with spectrophotometer)
- use two coupled enzyme reactions in one of which change can be recorded (ex absorbance)
- change [S] for in depth analysis
- Beer-Lambert law to convert absorbance units into molar conc. units
- Michaelis-Menten plot + Lineweaveer-Burke plot (to find out Vmax and Km)
What is pseudo order approximation used for?
Used for reactions with several substrates - two binding events - may / may not be ordered - each substrate has its own Km
What are the mechanism for enzyme catalysed reaction with two substrates?
- Ternary complex mechanism (Bi-Bi mechanism)
- Ping-pong mechanism (substituted enzyme mexhanism)
Explain ternary-complex mechanism of two substrate binding, explain using binding diagrams
In ternary-complex mechanism - both substrates have to bind to the enzyme for reaction to occur
A+B+E -> EAB -> EPQ -> E+P+Q
- EAB - ternary complex (two S and E)
- ordered binding of S and ordered release of P (binding of A allows binding of B - release of P allows release of Q - induced fit)
What is a Cleland diagram?
Cleland diagram - representation of ordered ternary-complex (Bi-Bi) enzyme mechanism
Explain Ping-Pong mechanism of two substrate binding
Ping-Pong mechanism - not sequential (A binds to E - forms new form of E (F) - P is released - B binds with new E form - another new E form - release Q) - enzyme is modified throughout the reaction
Are reaction rate equations the same for ternary-complex and ping-pong mechanisms?
No
How can the mechanism of a two substrate reaction be figured out using Lineweaver-Burke plot?
Plot several Lineweaver-Burke plots with varying [B]
- Ternary-complex: non-paralel graphs
- Ping-Pong: parallel graphs
