Enzyme Kinetics

Question 1

what is pre-steady state?

Answer 1

isolated enzyme mixed with substrate to build-up enzyme-substrate complex

steady state reached in microseconds

Question 2

why do enzymes exist in the steady state most of the time?

Answer 2

to regulate metabolism

Question 3

what are some assumptions of steady state?

Answer 3

• conc of enzyme [E] is negligible compared to substrate [S]

- no depletion in substrate pool, no accumulation in products —> v0 (initial velocity) = proportional to [E]0

Question 4

at a low [S] what is v0 proportional to?

Answer 4

[S]

Question 5

at a very high [S], what does v0 approach?

Answer 5

Vmax

Question 6

what is Km also known as?

Answer 6

Michaelis constant

Question 7

the formation of ES is what?

Answer 7

rapid and reversible

Question 8

what kind of a complex is an ES?

Answer 8

non-covalent

Question 9

what order kinetics will kcat follow?

Answer 9

1st order

Question 10

what is Ks?

Answer 10

equilibrium binding constant / association constant

Question 11

often Km is very close to Ks, what does this mean?

Answer 11

M-M kinetics are operable

Question 12

Km > Ks

what does this mean?

Answer 12

dissociation of ES = significant in comparison to Kcat (briggs-holdane kinetics)

Question 13

Km < Ks

what does this mean?

Answer 13

long lived intermediates exist after substrate binding

Question 14

what is the disadvantage of the lineweaver-burk plot?

Answer 14

• compression of data points with high [S] into small region
• this favours data with low [S]

Question 15

what are the advantages of the Eadie-Hofstee plot?

Answer 15

all values of [S] weighted equally

more accurate determination of Km

Question 16

what are the disadvantages of the Eadie-Hofstee plot?

Answer 16

time consuming

both axes depend on v0 introducing potential for experimental error

Question 17

what is the advantage of the Woolf-Hanes plot?

Answer 17

quicker to obtain data than E-H

Question 18

what is the disadvantage of the Woolf-Hanes plot?

Answer 18

Km = intercept not slope, so more prone to error than E-H

Question 19

what kind of complex is dihydrofolate reductase?

Answer 19

ternary

Question 20

what is the most common form of inhibition

and give example?

Answer 20

reversible

uses ethanol to treat methanol poisoning

inhibits alcohol dehydrogenase catalysed formation of formaldehyde, preventing blindness

Question 21

what is uncompetitive inhibition?

Answer 21

• inhibitor ONLY binds to ES complex
• stabilises complex -> harder for substrate to react + leave
• uncomp never seen in isolation

Question 22

where is mixed inhibition seen?

Answer 22

real life drug interactions

binds to E / ES

Question 23

give examples of uncomp/mixed inhibition?

Answer 23

• N-(phosphpnomethyl)glycine aka glycophosate
• uncomp inhibits 3-phosphoshikimate 3- carboxyvinyltransferase
• lithium ion treatment of manic depression -> uncomp inhibition of myo-inositol monophosphatase

Question 24

for comp inhibition, what will happen to Vmax?

Answer 24

unaffected

Question 25

for uncomp inhibition, what will happen to Km and Vmax?

Answer 25

altered

Question 26

irreversible inihibition?

Answer 26

• covalent bonding to/modification of active site
• permanent blocage of catalytic activity
• basis of nerve-agent attack upon serine proteases

Question 27

describe a LB plot of irreversible inhibition / non-comp inhibition?

Answer 27

• as inhibitor added active [E] drops

- this will dec Vmax but leave Km unchanged

Question 28

what time of sites to non-comp inhibitors bind to?

Answer 28

allosteric

diff shape