2.3 Enzyme kinetics

Question 1

Explain enzyme thermodynamics vs enzyme kinetics

Answer 1

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)

Question 2

What characteristics of a reaction needed to be able to investigate reaction kinetics?

Answer 2

Some kind of change that can be measured quantitatively:
- pH
- colour change
- mass change
- precipitate

Question 3

The basic reaction rate equation, what are the units

Answer 3

conc. / t
ex: mol dm-3 s-1; mg ml-1 min-1; molecules ml-1 min-1; Torr s-1

Question 4

How to figure out the rate at a specific time using the rate graph?

Answer 4

Rate at specific time - tangent at specific point - differential equation

Question 5

What is the relationship of reaction rate and stoichiometric equations?
O2 + 2H2 -> 2H2O

Answer 5

• Rate can be calculated from stoichiometric equations - ratios in which reactants / products are lost / produced
• Rate law CANNOT be calculated from stoichiometric equatinos!!

Question 6

What is the equation for rate determination from stoichiometric coefficient va?

Answer 6

aA + bB -> cC + dD
- rate of conc [A] against time t
- va : < 0 if reactants (add -) ; > 0 if products

Question 7

How are rate laws determined?

Answer 7

Rate laws for a specific reaction are only determined EXPERIMENTALLY

Question 8

What is the general rate law equation? Explain components

Answer 8

• 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

Question 9

What are the possible methods to solve >3 order equations?

Answer 9

When rate laws become >3 -> difficult to integrate:
- isolation method
- differentiation method

Question 10

Explain differential method to calculate reaction orders

Answer 10

• used when orders are >3: for enzyme reactions by changing substrate conc

Question 11

How does change in temperature affect reaction kinetics?

Answer 11

• 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

Question 12

Explain isolation method to calculate reaction orders

Answer 12

• 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 - ??

Question 13

Can rate laws be predicted by stoichiometric equations?

Answer 13

NO, rate laws are determined only experimentally

Question 14

Explain what is Arrhenius equation used for?

Answer 14

Arrhenius equation describes the effect of temperature on rate constant (k) and reaction rate

Question 15

What are Arrhenius parameters?

Answer 15

• A (pre-exponential factor)
• Ea

Question 16

Which graph is used to determine reaction Ea depending on T?

Answer 16

Question 17

What is a steady state approximation?

Answer 17

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

Question 18

What change in a reaction allows enzymes to act as catalysts? What are the mechanisms for the change?

Answer 18

Lowering activation energy:
- by binding a transition state - stabilising intermediate
- by changing the reaction pathway

Question 19

Which equation allows to account enzyme function: reaction rate change linking yo activation energy change

Answer 19

Arrhenius equation

Question 20

What graph is used ot depict enzyme catalysed reaction:
S + E -> ES -> E+P

Answer 20

Question 21

What does high/low Km represent?

Answer 21

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

Question 22

What is Kcat?

Answer 22

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

Question 23

How can enzyme efficiency be evaluated?

Answer 23

Enzyme efficiency = Kcat / Km

Question 24

What is Lineweaver-Burke plot? Why is it useful?

Answer 24

Lineweaver-Burke plot (linear equation) can be used to solve Michaelis-Menten equation

Question 25

What is the requirement for SSA application?

Answer 25

At the **start** of the reaction **[S] must be high** and** [P] low**

Question 26

How enzyme kinetics is determined in laboratory?

Answer 26

(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

Question 27

How coupled enzyme kinetics is determined in laboratory?

Answer 27

(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)

Question 28

What is pseudo order approximation used for?

Answer 28

Used for reactions **with several substrates** - two binding events - may / may not be ordered - **each substrate has its own Km**

Question 29

What are the mechanism for enzyme catalysed reaction with two substrates?

Answer 29

- **Ternary complex mechanism** (Bi-Bi mechanism) - **Ping-pong mechanism** (substituted enzyme mexhanism)

Question 30

Explain ternary-complex mechanism of two substrate binding, explain using binding diagrams

Answer 30

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)

Question 31

What is a Cleland diagram?

Answer 31

Cleland diagram - representation of ordered **ternary-complex** (Bi-Bi) enzyme mechanism

Question 32

Explain Ping-Pong mechanism of two substrate binding

Answer 32

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

Question 33

Are reaction rate equations the same for ternary-complex and ping-pong mechanisms?

Answer 33

No

Question 34

How can the mechanism of a two substrate reaction be figured out using Lineweaver-Burke plot?

Answer 34

Plot several Lineweaver-Burke plots with varying [B] - Ternary-complex: **non-paralel** graphs - Ping-Pong: **parallel** graphs