Enzyme Kinetics Flashcards
Describe enzymes
Catalysts: make reactions happen quickly
Work under mild physiological conditions
Specific for both reaction and substrate
Can be regulated
Describe general enzyme reaction equation
E + S (reversible reaction sign) ES = E + P
What does E stand for
Enzyme
What does S stand for
Substrate
What does P stand for
Product
What is enzyme kinetics
Study of enzyme reactions
Describe action of an enzyme (basic)
Enzyme substrate forms, is a reversible process
Product then dissociates and regenerates the enzyme
What happens with low levels of substrate
Only a small fraction of enzyme molecules can bind at any time
What happens at high substrate levels?
Most enzyme molecules substrate bound = saturated
When you measure enzyme kinetics what do you tend to be measuring?
Appearance of product or disappearance of substrate
Explain how you would measure enzyme activity of ß-galactose
ß-galactose is an enzyme that breaks down lactose to galactose and glucose
Give nitrophenylgalactose that breaks down to galactose and nitrophenolate which is yellow
Measure appearance of yellow colour over time
What is the rate of reaction?
Velocity of reaction
What does V stand for?
V
What is created when substrate concentration plotted?
Hyperbolic curve
-goes up then levels out over time
What is the Vmax
Maximum velocity a reaction can go
What is Km
The substrate concentration that produces the velocity of have the Vmax (not half the Vmax!!)
Tells us generally about affinity of the enzyme for the substrate
What is the michaelis-menten equation?
V = Vmax ([s]÷Km +[s])
V = velocity Vmax = max velocity Km = v= sub contraction at vmax ÷ 2 S = substrate concentration
What is k1
Binding rate
What is K2
Dissociation rate
What is Kcat
Catalytic constant (rate of production)
How do you work out velocity?
V = kcat x concentration of enzyme substrate complex
How do you mathematically describe enzyme catalysed reaction
Km = (k2+kcat) ÷ k1
How do you work out Vmax
Vmax = Kcat [Etot]
What is the conc ES
Same concentration as amount of enzyme put in
What is Etot
Total enzyme concentration (how much enzyme put in)
What is the relationship between Vmax and Etot
Directly proportional
E.g. 3rd less enzyme = Vmax a 3rd less
What is a specificity constant
How efficiently an enzyme converts substrate to product
Specificity constant equation
And how to tell from results if an enzymes specificity constant is good
Kcat/Km
Good enzyme:
Kcat = high
Km = low
Kcat/Km = high
How to work out Kcat
Vmax/ Etot
What is the lineweaver-Burke plot equation?
1/v = ((Km x 1)÷(Vmax x S)) + (1/Vmax)
Which sections of line weaver - Burke plot relate to equation of straight line
(Equation of straight line: y = Mx + C)
1/v = y Km/Vmax = m 1/s = x 1/vmax = c
What does c stand for in straight line equation
Intercept
How do you work out slope of line from line weaver Burke plot
Km/vmax
What are the uses of enzyme inhibition?
Understanding how enzymes work
Producing drugs
What are transition states
Help pull substrate into structure that lowers energy barrier for a reaction
Competitive inhibition
- inhibitor similar to substrate so binds to enzymes active site
- competitive inhibition because substrate and inhibitor compete to bind
- reversible so inhibitor will dissociate after time and then substrate gets a chance to bind
E + I (reversible reaction sign) EI
How can you recognise competitive inhibition in enzyme kinetics
V max doesn’t change after inhibitor (if loads of substrate then inhibitor wont make much difference)
There’s an apparent change in Km but no actual change
What is the equation for Kmapp (the apparent Km)
Km app = Km (1 + I/Ki)
What is Ki
Inhibitor constant (affinity of inhibitor to enzyme)
The lower the value the best tier = more effective at lower amount
Irreversible reaction
- inhibitors that react covalently with groups within enzymes that are essential to enzymes activity
- irreversible
E + I = EI
How to see irreversible inhibition through enzyme kinetics
Vmax changes as enzymes inactivated permanently
Concentration of substrate needed to reach vmax stays the same
Km stays the same
