Lecture 6

Question 1

What is binding free energy

Answer 1

The difference between the activation energy of the uncatalysed reaction and that of the catalysed reaction

Question 2

What is Gcat

Answer 2

net of Guncat and binding energy

Question 3

What are the enzyme kinetics about pH

Answer 3

• pH:

* at extremes of pH, most enzymes are denatured, although some continue to function under: phosphatase, pH optimum 9.5

Question 4

What are the enzyme kinetics about temperature

Answer 4

• Temperature:

* rate of reaction increases with temperature, e.g. from 0 C to 37 C, then decreases as the protein is denatured

Question 5

What are the enzyme kinetics about concentration of substrate(s)

Answer 5

• as substrate concentration increases, the rate of reaction increases

Question 6

What are enzyme kinetics about The presence of inhibitors of the enzyme

Answer 6

inhibitors bind the enzyme, either at the active site, or elsewhere on the enzyme, to cause changes
• binding can be reversible or irreversible

Question 7

What are the enzyme kinetics about presence of activators of the enzyme

Answer 7

bind the enzyme at the active site or elsewhere and regulate activity
by increasing the rate of reaction

Question 8

Enzyme kinetics is described in terms of

Answer 8

• The initial rate of an enzyme‐catalysed reaction, Vo
• The corresponding concentration of substrate, [S]
• The maximum rate of reaction for a given enzyme, Vmax
• The degree of saturation of an enzyme by its substrate

Question 9

What is the activity of a enzyme defined as

Answer 9

rate of appearance of product P or rate of disappearance of substrate S (initial velocity or Vo) as time approaches zero

Question 10

What does the measurement of Vo at a series of [S] do

Answer 10

used to estimate Vmax and Km -michaelis-Menten kinetics

Question 11

What are the steps to measure the kinetic properties of the enzyme glucose 6‐
phosphate dehydrogenase (G6PD or G6PDH)

Answer 11

• Setting up a series of reaction mixtures in which the substrate concentration, [S], is the only variable
• Using a spectrophotometric method to follow the production of NADH (from reduction of the substrate NAD+) over time
• Determining the initial rate of the reaction (Vo) at each [S]
• Handling the data for [S] and the associated Vo by two graphical methods to determine Km and Vmax

Question 12

DETERMINATION OF INITIAL VELOCITY,

V0

Answer 12

1. Set up a reaction with all
   components needed except the enzyme or a substrate
2. Start the reaction by the addition of enzyme or substrate
3. Follow the production of P or removal of S over time
4. From a plot of [P] versus time, draw tangent to curve near time
   zero and measure slope of the curve to give initial velocity, Vo

Question 13

Waht is the Vo defined as

Answer 13

Vo = Vmax[S]/(Km+[S])

Question 14

What are the properties of Lineweaver-Burk plot(double reciprocal)

Answer 14

1/Vo versus 1/[S]

1/Vo = 1/Vmax + Km/Vmax[S]

Straight line of the form: y= b +ax
With a slope of Km/Vmax
intercept on the y axis of 1/Vmax
intercept on the x axis is -1/Km

Question 15

GLUT isoforms differ in their

Answer 15

• Tissue expression
• Substrate specificity
• kinetic characteristics

Question 16

KINETIC PROPERTIES of GLUT 2

Answer 16

Regulates insulin release in
pancreas, removes excess
glucose from blood in liver

Tissues of liver and beta cells

low affinity for glucose
Km : 15-20mM

Question 17

KINETIC PROPERTIES of GLUT 4

Answer 17

increases with endurance training, insulin increases activity

tissues of Muscle, fat, heart

Km: 5mM

Responses to small increase or decrease of glucose

Question 18

What is the function of hexokinase I from muscles

Answer 18

e (Km about 0.1 mM) acts at a maximal rate unless reaction is inhibited by accumulation and inhibits the reaction

Question 19

What is thhe function of hexokinase IV from liver

Answer 19

(Km about 10mM) can continue to respond as the concentration of glucose increases