Enzyme kinetics

Question 1

How do enzymes lower the activation energy?

Answer 1

1. enzyme binding of substrate = reduction of freedom of movement
2. enzymes distort the substrate towards the transition state
3. multiple weak bonds between AS + S can offset activation energy
4. provide alternate reaction path

Question 2

Specificity is determined by…

Answer 2

interactions with amino acid side chains within the co-enzyme binding pocket
~ hydrophobic, electrostatic interactions or H bonds

Question 3

lactate dehydrogenase

Answer 3

~ tetramer

~ NAD cofactor binds to co-enzyme binding pocket

Question 4

Why is enzyme kinetics useful?

Answer 4

~ cost-effective was of understanding mechanisms of enzyme-catalysed reactions
~ estimations of binding affinities of enzymes (substrate/inhibitor)
~ understandin enzyme properties for design of inhibitors or clinical assays

Question 5

Name two enzymes who have clinal assays that rely on a measure of their activity.

Answer 5

~ aspartate transaminase

~ alanine aminotransferase

Question 6

‘standard’ assays measure

Answer 6

initial velocity = before 10% of S has been consumed

Question 7

Name two methods of measuring P appearance or S disappearance

Answer 7

~ spectrophotometrically

~ isolate isotope-enriched S/P using chromatography

Question 8

When is a coupled assay used?

Answer 8

when it is not possible to measure substrate utilisation or product appearance directly

Question 9

What is a coupled assay?

Answer 9

~ use P from the reaction you wish to study as S for another reaction
~ properties of 2nd S/P need to be able to be followed as a function of time

Question 10

Steady state kinetics requires 2 assumptions about what happens when enzyme and substrate are mixed, what are they?

Answer 10

1. assumption of equilibrium

2. assumption of steady state

Question 11

Michaelis-Menten equation states…

Answer 11

the rate of an enzyme catalysed reaction is proportional tot he amount of the enzyme-substrate complex

Question 12

What happens when substrates saturates and entirely converts E to ES?

Answer 12

~ 2nd step becomes rate-limiting

~ rate is insensitive to changes in substrate

Question 13

In a plot of initial velocity, K(M)…

Answer 13

~ is the substrate concentration at which the reaction velocity is half/maximal (1/2 Vmax)
~ varies as a function of temperature and pH

Question 14

In a plot of initial velocity, K(M)…

Answer 14

~ the maximal reaction velocity

~ where the graph plateaus

Question 15

How does one draw a Lineweaver-Burk plot?

Answer 15

~ straight line
~ +ve and -ve X axis
~ 1/Vmax = (X=0)
~ -1//Km = (Y=0)

Question 16

K(cat)

Answer 16

a.k.a. turnover number

~ a measure of catalytic efficiency

Question 17

Diffusion-controlled limit

Answer 17

~ for frequency of A and S collisions

~ range of: 10^8-10^9 M(-1)s(-1)

Question 18

Circe effect

Answer 18

~ using strong attractive force to lure S into AS

~ an explanation for catalytic perfection