P4 - Enzyme Kinetics of LDH

Question 1

what is the steady state assumption in the Michaelis Menten equation?

Answer 1

the rate of ES production is equal to the overall rate of the reactions that decrease [ES] (k1=k-1)

Question 2

what is Km?

Answer 2

substrate concentration at which the reation rate is half the maximal rate (1/2Vmax)

Question 3

what does it mean if an enzyme has a low Km?

Answer 3

it achieves maximal catalytic efficiency at low [S]

Question 4

what is kcat?

Answer 4

the turnover number
the number of reaction processes that each active site catalyses per unit time

Question 5

what are disadvantages of lineweaver-burk plot?

Answer 5

-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

Question 6

what is a competitive inhibitor?

Answer 6

-molecule that resembles substrate structure
-competes directly with substrate for active site
-binds to active site but is unreactive

Question 7

what parameters are affected by competitive inhibition?

Answer 7

Km (affinity for substrate binding)
Vmax is not affected

Question 8

what is an uncompetitive inhibitor?

Answer 8

• molecule that binds ro an allosteric site on ES complex
• can cause distortion of active site

Question 9

what parameters are affected by uncompetitive inhibition?

Answer 9

Vmax
Km

Question 10

what is a mixed inhibitor?

Answer 10

-molecule that binds to allosteric site
-can bind to free enzyme or ES complex
-effective inhibition at both low and high [S]

Question 11

what parameters are affected by mixed inhibition?

Answer 11

Vmax
Km

Question 12

how is competitive inhibition overcome?

Answer 12

high concentrations of substrate

Question 13

how is uncompetitive inhibition overcome?

Answer 13

lowering substrate concentration

Question 14

what are the features of a perfectly evolved enzyme?

Answer 14

diffusion controlled
Km much higher than physiological [S]

Question 15

where is the true initial rate on a graph?

Answer 15

where the rate of reaction is proportional to enzyme concentration, at the start of the reaction

Question 16

what happens [ES] after increased concentration of substrate?

Answer 16

it does not change

Question 17

what is a first order reaction?

Answer 17

rate of reaction depends on concentration of reactant

Question 18

what is occuring at Vmax in relation to [S]?

Answer 18

all substrate is being made into a product

Question 19

how do you identify Vmax and Km on Lineweaver Burk?

Answer 19

intercept on y-axis = 1/Vmax
intercept on x-axis = -1/Km

Question 20

how many substrates does LDH bind and by what mechanism?

Answer 20

2, sequentially (one first and then the other)

Question 21

what happens pyruvate and NADH in LDH active site?

Answer 21

pyruvate is reduced, NADH is oxidised

Question 22

what is the Michaelis Menten equation when considering a sequential reaction?

Answer 22

Vo= Vmax/1+Km(A)/[A] + Km(B)/[B] + Ks(A)Km[B]/[A][B]

Question 23

What happens a reaction when enzyme concentration increases?

Answer 23

it should increase proportionally

Question 24

if enzyme concentration is 10uM and the reaction rate is 0.35A/min, what should it be at 20uM?

Answer 24

0.7A/min

Question 25

how is LDH activity recognised?

Answer 25

decrease in absorption/ NADH

Question 26

why does increasing pH of solution, drive LDH catalysed reaction to NAD reduction (NADH production)?

Answer 26

the equilibrium shifts to the left - Le Chatelier’s Principle

Question 27

4what happens reaction with constant LDH concentration and varying pyruvate concentration?

Answer 27

the rate of reaction increased as substrate concentration increased

Question 28

What does LDH catalyse in anaerobic conditions?

Answer 28

reduction of pyruvate to lactate
last step of glycolysis

Question 29

what 2 subunits make up LDH?

Answer 29

M and H

Question 30

How many tetrameric isoenzymes does LDH have?

Answer 30

5

Question 31

what is the final concentration of lactate in the following solution:
2.7mL 100mM SodPhos
0.2mL 900mM sodium lactate
0.1mL 180mM NAD

Answer 31

Sv X Sc = Fv X Fc
0.2 X 900 = 3 X Fc
= 60mM

Question 32

why does lactate and NAD have aimilar Vmax values?

Answer 32

no substrate inhibition
sudden release of energy needed anaerobically - this requires both

Question 33

how do you obtain true Vmax and Km?

Answer 33

by plotting 1/Vo against 1/[lactate/NAD]

Question 34

what type of reaction is LDH-M4 catalysis?

Answer 34

near equilibrium

Question 35

what happens when you rasie pH in a near-equilibrium reaction?

Answer 35

the reaction shifts towards the left (more reactants) Le Chatelier’s Principle - reestablishing equilibrium

Question 36

what does km mean?

Answer 36

a measure of enzyme affinity for a particular substrate

Question 37

what is vmax?

Answer 37

maximum initial velocity of a reaction

Question 38

what is plotted on michaelis menten graph?

Answer 38

initial rate V vs substrate concentration

Question 39

what is Km equal to?

Answer 39

(k2+k-1)/k1

Question 40

what is the michaelis menten equation?

Answer 40

V0 = Vmax[S]/Km + [S]

Question 41

what is the lineweaver burke equation?

Answer 41

1/V0 = Km/Vmax[S] + 1/Vmax

Question 42

what is the slope in wine weaver burke plot?

Answer 42

Km/Vmax

Question 43

do enzymes alter over all change in free energy of a reaction?

Answer 43

no

Question 44

do enzymes alter over all change in free energy of a reaction?

Answer 44

no

Question 45

do enzymes speed up reactions by raising Km?

Answer 45

no