Enzyme Kinetics Flashcards
What is enzyme kinetics?
The study of the rate of an enzyme-catalysed reaction and how that rate varies with different conditions
What is the rate of a reaction?
Decrease in substrate concentration/increase in product concentration
Per unit time
Describe the rate-concentration graph of a typical enzyme with saturation hyperbolic kinetics
Michaelis-Menten
Low substrate concentration = linear/first order as rate is proportional to [substrate]
High substrate concentration= zero order as rate is independent of [substrate]
What is Vmax?
The maximum velocity of a reaction that occurs at infinite substrate concentration
What is the Michaelis-Menten reaction model?
E + S —> ES —> E + P
With rate constants
What assumptions do we make when using the Michaelis-Menten reaction model?
[S] is much greater than [E] so only a small amount of substrate bound at any one time
[ES] does not change with time (formation = breakdown into E+S/P) - steady state approximation
Initial velocities used so [P] is small so E + P —> ES can be ignored (k-2)
What equation is used to find the rate of a reaction?
V0 = (Vmax.[S]) / (Km+[S])
What does the substrate concentration represent when V0 = Vmax/2?
Km
How do you calculate Km using rate constants?
Km = (K2 + K-1) / K1
When is Km approximately the Kd for ES?
When K2 is very small/rate-limiting
What does Km represent when K2 is rate-limiting?
Measurement of affinity of enzyme for its substrate in the ES complex (Kd)
What does a low Km mean?
High affinity of enzyme for substrate
What is Kcat?
Turnover number
Number of substrate molecules converted to product in a given unit of time on a single enzyme molecule when saturated with substrate
What is the equation relating Vmax, Kcat and [E]t?
Vmax = Kcat.[E]t
What does a high Kcat mean?
Reaction can occur faster (higher Vmax)
What is the specificity constant?
Number used to quantify catalytic efficiency of enzymes
How do you calculate the specificity constant?
Specificity constant = Kcat / Km
What does a high specificity constant show?
Enzyme is very efficient
What is the Lineweaver-Burk equation?
1/V0 = Km/Vmax . 1/[S] + 1/Vmax
What are on the axes of a Michaelis-Menten graph?
X = [S]
Y = initial reaction velocity
What are on the axes of a Lineweaver-Burk plot?
X = 1/[S]
Y = 1/V0
How do you find 1/Vmax on a Lineweaver-Burk plot?
Y-intercept
How do you find -1/Km on a Lineweaver-Burk plot?
X-intercept
What does the y-intercept represent on a Lineweaver-Burk plot?
1/Vmax
What does the x-intercept represent on a Lineweaver-Burk plot?
-1/Km
How can you calculate the gradient of a Lineweaver-Burk plot?
Km/Vmax
What is an example of a competitive inhibitor?
Malonate inhibiting succinate dehydrogenase
How does a competitive inhibitor work?
Similar chemical structure to substrate
Binds to active site of enzyme but does not activate/react
What value does a competitive inhibitor affect?
Km = [S] must increase to outcompete inhibitor
Vmax unaffected as inhibitor can be surmounted
What value does a non-competitive inhibitor affect?
Vmax = turnover of substrate prevented and effect cannot be surmounted
(Km unaffected as inhibitor does not affect affinity of enzyme for its substrate)
What do ACE inhibitors do in the body?
Inhibit ACE (angiotensin converting enzyme)
So no angiotensin II produced
So no periphery vasoconstriction
Blood pressure does not increase
What is an example of an ACE inhibitor?
Captopril
What is a competitive reversible inhibitor of acetylcholine-esterase?
Neostigmine
What is a competitive irreversible inhibitor of acetylcholine-esterase?
Diisopropyl fluorophosphate/dyflos
How does neostigmine work?
Competitive reversible inhibitor of acetylcholine-esterase
Forms covalent bond with Ser (of catalytic triad)
Bond is hydrolysed over time
How does diisopropyl fluorophosphate work?
Competitive irreversible inhibitor of acetylcholine-esterase
Bonds covalently with no spontaneous hydrolysis
What can be used to reactivate AChE after using diisopropyl fluorophosphate?
Pralidoxime
What are neostigmine and diisopropyl fluorophosphate used to treat?
Alzheimer’s disease
How can enzyme activity be regulated?
Allosteric regulators with allosteric enzymes
Covalent modification by other enzymes
Induction/repression of enzyme synthesis
What kind of graph represents the enzyme activity of an allosteric enzyme?
Sigmoid
What is an example of allosteric inhibition?
ATP and citrate on phosphofructokinase
What is an example of allosteric activation?
Phosphoenol pyruvate (PEP) and fructose-1,6-bisphosphate on pyruvate kinase
What residues are usually (de)phosphorylated in covalent modifications?
Ser
Thr
Tyr
His
How is covalent modification used to activate and inactivate glycogen phosphorylase?
Phosphorylase kinase phosphorylates Ser 14 using ATP to activate
Phosphorylase phosphatase dephosphorylates Ser 14 to inhibit
What are some types of covalent modification of enzymes?
Phosphorylation
Adenylylation/Uridylylation of Tyr
ADP ribosylation (Arg, Gln, Cys)
Methylation (Glu)
Acetylation (Lys)
What happens in adenylylation/uridylylation?
ATP or UDP —> PPi
Tyrosine residues
What happens in ADP ribosylation?
Arg, Gln, Cys
NAD —> nicotinamide
What happens in methylation?
Glu
S-adenosyl-methionine —> S-adenosyl-homocysteine
What happens in acetylation?
Lys on histones
Increased transcription
What is an example of induction of enzyme synthesis?
High blood glucose stimulates insulin release
Insulin increases rate of synthesis of key enzymes involved in glucose metabolism (glucokinase, phosphofructokinase, pyruvate kinase)
