Energetics and dynamics of protein action

Question 1

Hydrolase

Answer 1

hydrolytic cleavages

Question 2

polymerase

Answer 2

polymerisation reactions

Question 3

synthase

Answer 3

synthesis

Question 4

kinase/phosphatases

Answer 4

add/remove phosphates

Question 5

isomerases

Answer 5

rearrangements

Question 6

oxide-reductases

Answer 6

oxidise/reduce substrates

Question 7

ATPases

Answer 7

use ATP

Question 8

What is BRENDA?

Answer 8

a database that tells you about proteins

Question 9

What are the stages of an enzyme substrate reaction?

Answer 9

S+E=>ES=>EP=>E+P

Question 10

What is an endergonic reaction?

Answer 10

When ΔG is greater than 0

Question 11

What is an exergonic reaction?

Answer 11

When ΔG is less than 0

Question 12

What reactions tend to be spontaneous?

Answer 12

exergonic

Question 13

How can you make an endergonic reaction spontaneous?

Answer 13

by coupling to with a highly exergonic reaction

Question 14

What is the induced fit model?

Answer 14

proposes distortion of enzyme and substrate is important in catalysis

Question 15

What is catalysis dependant on?

Answer 15

• localisation of substrate
• orientation of substrate
• binding energy of substrate
• catalytic residues on protein framework

Question 16

What is the activation energy?

Answer 16

the energy required to reach the transition state (bent stick)

Question 17

When a protein binds to an enzyme why does ΔG initially fall?

Answer 17

because the enzyme will displace some water molecules which will increase disorder

Question 18

What does the enzyme need to be complimentary to?

Answer 18

the transition site

Question 19

How can a structural change provide activation energy?

Answer 19

in hexokinase, when in open form it is unlikely to react. When glucose binds it changes its conformation to closed form which allows ATP to bind.

Question 20

How can conformational changes affect ΔG?

Answer 20

allow compensation between ΔH and ΔS to minimise ΔG

Question 21

What is K2?

Answer 21

ES => E + P

Kcat

Question 22

What is Kcat?

Answer 22

turnover number of the enzyme (how good it is) (rate that ES is converted to E + P

Question 23

What is the steady state assumption?

Answer 23

the concentration of ES stays the same (rate of formation of ES=rate of degradation of ES)

Question 24

What are the assumptions of M-M scheme?

Answer 24

1. assume that the reverse reaction is negligible (while its favourable we can establish experimental conditions that preclude or minimise the reverse reaction)
2. assume only a single central complex (ES) exists (ES breaks down directly to +P
3. Assume that [S}&raquo_space;[E] interaction of S with E does not significantly affect the concentration of S

Question 25

What is Vmax?

Answer 25

the max velocity that can enzyme can work at

Question 26

What is the equation for Vmax

Answer 26

Vmax=Kcat[E]

Question 27

What is the Km?

Answer 27

the substrate conc when the reaction rate is half maximal (michaelis constant) (measure of affinity of the enzyme for the substrate)

Question 28

What is the Michaelis menten equation?

Answer 28

velocity = Vmax[S] ------------- Km+[S]

Question 29

How do you measure enzyme efficiency?

Answer 29

cat/kM

Question 30

Why do you have to be careful when using Michaelis-Menton?

Answer 30

- does not explain kinetic properties of many enzymes | - example of sigmoidal curves for proteins with multiple subunits such as allosteric enzymes

Question 31

What is the upper limit of enzyme efficiency and why is there an upper limit?

Answer 31

1x10^8M^-1.s^-1 limited by time taken for substrate to diffuse into active site

Question 32

What is a problem of the michaelis menten equation?

Answer 32

you can't work out Vmax because infinite amounts of substrate are needed

Question 33

How do you get the Lineweaver-Burk equation?

Answer 33

by flipping the michaelis-menton equation over (which changes michealis-menten equation into a straight line)

Question 34

What are the components of the y=mx+c equation?

Answer 34

y= 1/v0 m=km/Vmax x= 1/[S] c= 1/Vmax

Question 35

What is the x intercept in a Lineweaver-Burk plot?

Answer 35

-1/Km

Question 36

What is the y intercept?

Answer 36

1/Vmax

Question 37

What is the limitation of the Lineweaver-Burk plot?

Answer 37

its highly sensitive to measurements at low [S]

Question 38

If it is 1/v 1/[S] graph where is high substrate conc?

Answer 38

At the bottom close to both axis

Question 39

What happens in competitive inhibition?

Answer 39

- Km increases because less substrate is binding so affinity is lower - Vmax is unaltered because if you increase [S] you can outcompete the inhibitor

Question 40

How do non-competitive inhibitors work?

Answer 40

- molecule binds to site on enzyme other than the active site - modifies the enzyme conformation to slow/prevent product formation - high affinity for second binding site

Question 41

What happens to the Km of non-competitive inhibition?

Answer 41

- Km is unaltered (substrate can still bind but can't be converted as quickly into products Tham without the inhibitor) - Vmax is reduced (product formation slows down

Question 42

Give one example of naturally occurring non-competitive inhibitors?

Answer 42

-caffeine

Question 43

Give an example of a synthetic non-competitive inhibit?

Answer 43

haloperidol

Question 44

Give an example of a naturally occurring competitive inhibitor?

Answer 44

digitalis

Question 45

Give an example of a synthetic competitive inhibitor?

Answer 45

ibuprofen

Question 46

Explain why non-competitive inhibition is common in feedback inhibition, using threonine as an example.

Answer 46

- biosynthesis of isoleucine from threonine in bacteria involves 4 steps and 4 enzymes - first reaction is catalysed by threonine deaminase - this reaction is noncompetitvely inhibited by isoleucine, the end product of the pathway - as the product increases the first reaction rate decreases - this leads to less product and a reduction in the inhibition as the isoleucine dissociates from the enzyme - the cycle begins again