Proteins and enzymes, Section 5, enzyme kinetics (Dr. Taylorson)

Question 1

What is a reaction rate ?

Answer 1

It is the the amount of product formed per unit of time.

Question 2

Why do we only consider V zero (initial velocity) for enzymes ?

Answer 2

Because this value is more precise. After a while, reverse reactions and inhibition factors come into play and these alter our measurement.

Question 3

What is the simplest way to investigate reaction rate ?

Answer 3

By monitoring the increase in product against time. This can be done at a variety of substrate concentration and the initial velocity of the reaction is measured.

Question 4

What is the general formula for an enzyme reaction ?

Answer 4

E + S –> E.S –> E + P w/ k+1 and k+2 as rate constants (and k-1 and k-2 respectively).

Question 5

What are the assumptions to measure the rate at which product is formed ?

Answer 5

At the initial velocity, [P] = 0 and [S]»[E]

This means that, since there is so much more substrate, the amount of E.S is constant and thus only P is formed.

Question 6

What is the formula to calculate V zero ?

Answer 6

V zero = Vmax.[S} / [S] + Km

Question 7

What is Km (or the Michaelis constant) ?

Answer 7

It is the concentration of substrate that half-saturates the enzyme’s active site.

Question 8

We’ve plotted V zero against [S].

What is the shape of the curve ?

Answer 8

A rectangular hyperbola.

Question 9

What does Km measure ?

Answer 9

The affinity of the enzyme for its substrate. (it is like Kd for proteins binding ligands).
A low Km means a high affinity of the enzyme for its substrate.

Question 10

What does Vmax depend on ?

Answer 10

The initial concentration of enzyme

Question 11

What is Kcat ?

Answer 11

It is the turnover number, i.e. the of substrate molecules transformed per molecules of enzyme per second (units = reciprocal seconds).

Question 12

How can we calculate Kcat ?

Answer 12

Kcat = Vmax / [E(zero)]

Question 13

What is the specificity constant ?

Answer 13

Specificity constant = Kcat/Km

It is a measure of the efficiency of the enzyme.

Question 14

What is the Lineweaver-Burk Plot (or double reciprocal plot) ?

Answer 14

1/V(zero) = (Km/Vmax)*(1/[s]) + (1/Vmax)

Question 15

What are the the two types en enzyme inhibitions ?

Answer 15

Reversible and irreversible.

Question 16

What are the 3 types of enzyme inhibitors ?

Answer 16

Competitive, uncompetitive and non-competitive.
Generally (very generally!) competitive = reversible
uncompetitive = irreversible
non-competitive = irreversible

Question 17

Where to the 3 types of inhibitors bind ?

Answer 17

Competitive inhibitors binds to the enzyme’s AS, thus increasing the Km of the enzyme (Vmax is unaffected because if we saturate the system w/ substrate it will compete out the inhibitor)
Uncompetitive inhibitors bind the enzyme in the AS or not, and decrease both the Vmax and the Km of the enzyme. These are however really rare.
Non-competitive inhibitors bind the enzyme in place distinct from the AS and change the shape of the enzyme (and thus that of the AS) decreasing the Vmax of the enzyme (Km is unaffected because the substrate can still bind).