L14 - enzyme substrate interactions

Question 1

outlines

Answer 1

• how simple enzymes behave;
• how simple models describe enzyme kinetic behaviour
• how to quantify enzyme kinetic behaviour
• meaning of kinetic parameters
• derive numerical values for the various kinetic constants.

Question 2

what do enzyme kinetics tell us?

Answer 2

how fast reactions occur

Question 3

why is knowing enzyme kinetics important?

Answer 3

it can explain biological (disease) processes and explains what goes wrong in diseases

Question 4

what does enzyme kinetics describe?

Answer 4

absorbance of drugs into cells by active transport (pharmaceutics)

Question 5

why do we study enzyme kinetics?

Answer 5

1. scientific reasons
2. clinical tests can depend on measuring activity of enzymes
3. drug development: many drugs are enzyme inhibitors and enzyme kinetics can tell us the mechanism of a reaction and allows us to determine drug potency

Question 6

factors that affect enzyme reactivity

Answer 6

1. pH: changes ionsation of side chains on and within enzyme or change in ionisation of substrate
2. denaturing agents (e.g detergents, urea)
3. temp: starts of by increasing rate but then thermal denaturation occurs (occurs at 4 degrees above normal temp)

Question 7

what does the amount of product produced in a reaction need to be proportional to?

Answer 7

the amount of enzyme

Question 8

is there usually more enzyme or substrate in a reaction?

Answer 8

substrate conc is usually higher as the enzyme is a catalyst so is reused

Question 9

what do simple models of enzyme behaviour assume

Answer 9

1. only ONE molecule of ONE substrate binds to ONE SPECIFIC enzyme
2. that the enzyme + substrate will form an ESC (rate limiting/slow step)
3. that the enzyme converts substrate into product and that product release occurs (fast) (this is often not case as it can be rate limiting step)
4. products bind weakly to enzymes

Question 10

why are one substrate reactions often rare?

Answer 10

due to isomerism and rearrangements

Question 11

michaels menten equation

Answer 11

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

Question 12

what does v stand for?

Answer 12

rate

Question 13

what does [S] stand for?

Answer 13

substrate conc

Question 14

what does Vmax stand for?

Answer 14

rate when all enzyme active sites are occupied by sustrates (saturated enzymes). limiting rate.

Question 15

what does Km stand for?

Answer 15

substrate concentration when the rate is half vmax (the max rate)

Question 16

describe the rate when the [S] is low

Answer 16

at low substrate concentration the rate increases with increasing substrate concentration so it is first order

Question 17

describe the rate when the [S] is high

Answer 17

at a high substrate concentration the rate does not increase as substrate conc increased so zero order

Question 18

what does the michaelis menten plot need to look like?

Answer 18

1. v on y axis.
2. substrate conc (mM) on x axis
3. requires computer programme

Question 19

what does the lineweaver burk plot look like?

Answer 19

plot 1/v over 1/[S]

• y intercept is 1/vmax
• x intercept is -1/km

this graph gives HIGHER precision but lower accuracy due to difference of errors bars in different parts of graph and the least squares regression is not appropriate, don’t use excel

Question 20

what does the direct linear plot look like?

Answer 20

• plot rate on y axis and [S] onto the minus x axis
• connect points
• intersections are estimates of Km and Vmax
• gives median value
• no assumptions made about erros
• difficult deviations from ideal behaviour

Question 21

what is km often similar to?

Answer 21

[S] in cell

Question 22

what units could vmax be?

Answer 22

nmoles (nanomoles) product/min

Question 23

what is Kcat?

Answer 23

catalytic constant. first order rate constant for conversion of substrate to product. so Kcat =k3

Question 24

how do you calculate Kcat?

Answer 24

vmax / amount of enzyme (nmoles)