P4 - Enzyme Kinetics of LDH Flashcards
what is the steady state assumption in the Michaelis Menten equation?
the rate of ES production is equal to the overall rate of the reactions that decrease [ES] (k1=k-1)
what is Km?
substrate concentration at which the reation rate is half the maximal rate (1/2Vmax)
what does it mean if an enzyme has a low Km?
it achieves maximal catalytic efficiency at low [S]
what is kcat?
the turnover number
the number of reaction processes that each active site catalyses per unit time
what are disadvantages of lineweaver-burk plot?
-1/[S] values end up crowded on y-axis, drawing straight line can be inaccurate
-Fot small [S], there can be large errors in Km and Vmax
what is a competitive inhibitor?
-molecule that resembles substrate structure
-competes directly with substrate for active site
-binds to active site but is unreactive
what parameters are affected by competitive inhibition?
Km (affinity for substrate binding)
Vmax is not affected
what is an uncompetitive inhibitor?
- molecule that binds ro an allosteric site on ES complex
- can cause distortion of active site
what parameters are affected by uncompetitive inhibition?
Vmax
Km
what is a mixed inhibitor?
-molecule that binds to allosteric site
-can bind to free enzyme or ES complex
-effective inhibition at both low and high [S]
what parameters are affected by mixed inhibition?
Vmax
Km
how is competitive inhibition overcome?
high concentrations of substrate
how is uncompetitive inhibition overcome?
lowering substrate concentration
what are the features of a perfectly evolved enzyme?
diffusion controlled
Km much higher than physiological [S]
where is the true initial rate on a graph?
where the rate of reaction is proportional to enzyme concentration, at the start of the reaction
what happens [ES] after increased concentration of substrate?
it does not change
what is a first order reaction?
rate of reaction depends on concentration of reactant
what is occuring at Vmax in relation to [S]?
all substrate is being made into a product
how do you identify Vmax and Km on Lineweaver Burk?
intercept on y-axis = 1/Vmax
intercept on x-axis = -1/Km
how many substrates does LDH bind and by what mechanism?
2, sequentially (one first and then the other)
what happens pyruvate and NADH in LDH active site?
pyruvate is reduced, NADH is oxidised
what is the Michaelis Menten equation when considering a sequential reaction?
Vo= Vmax/1+Km(A)/[A] + Km(B)/[B] + Ks(A)Km[B]/[A][B]
What happens a reaction when enzyme concentration increases?
it should increase proportionally
if enzyme concentration is 10uM and the reaction rate is 0.35A/min, what should it be at 20uM?
0.7A/min
how is LDH activity recognised?
decrease in absorption/ NADH
why does increasing pH of solution, drive LDH catalysed reaction to NAD reduction (NADH production)?
the equilibrium shifts to the left - Le Chatelier’s Principle
4what happens reaction with constant LDH concentration and varying pyruvate concentration?
the rate of reaction increased as substrate concentration increased
What does LDH catalyse in anaerobic conditions?
reduction of pyruvate to lactate
last step of glycolysis
what 2 subunits make up LDH?
M and H
How many tetrameric isoenzymes does LDH have?
5
what is the final concentration of lactate in the following solution:
2.7mL 100mM SodPhos
0.2mL 900mM sodium lactate
0.1mL 180mM NAD
Sv X Sc = Fv X Fc
0.2 X 900 = 3 X Fc
= 60mM
why does lactate and NAD have aimilar Vmax values?
no substrate inhibition
sudden release of energy needed anaerobically - this requires both
how do you obtain true Vmax and Km?
by plotting 1/Vo against 1/[lactate/NAD]
what type of reaction is LDH-M4 catalysis?
near equilibrium
what happens when you rasie pH in a near-equilibrium reaction?
the reaction shifts towards the left (more reactants) Le Chatelier’s Principle - reestablishing equilibrium
what does km mean?
a measure of enzyme affinity for a particular substrate
what is vmax?
maximum initial velocity of a reaction
what is plotted on michaelis menten graph?
initial rate V vs substrate concentration
what is Km equal to?
(k2+k-1)/k1
what is the michaelis menten equation?
V0 = Vmax[S]/Km + [S]
what is the lineweaver burke equation?
1/V0 = Km/Vmax[S] + 1/Vmax
what is the slope in wine weaver burke plot?
Km/Vmax
do enzymes alter over all change in free energy of a reaction?
no
do enzymes alter over all change in free energy of a reaction?
no
do enzymes speed up reactions by raising Km?
no
