FIII: Enzyme Kinetics (Slides 20 - 67)

Question 1

What do enzymes do?

Answer 1

Speed up the rate of reaction proportionally to the amount of enzyme that is present.

Question 2

Enzyme Assay

Answer 2

The process of measuring enzyme catalyzed reaction rates

Question 3

Enzyme Kinetics

Answer 3

The mathematical analysis on how rate varies when you increase substrate concentration, but keep enzyme concentration constant.

Question 4

What happens when an enzyme acts on a substrate?

Answer 4

The substrate decreases and the product increases.

Question 5

What would chemists use instead of the real enzyme reaction products?

Answer 5

Direct analysis of enzyme reaction products can be time consuming - a better way is using artificial substrates

Question 6

What is an artificial substrate?

Answer 6

It is a molecular “look alike” for the real substrate

Question 7

What does Trypsin hydrolyze?

Answer 7

Trypsin hydrolyzes the C-terminal of Arg and Lys in a peptide

Question 8

How can you measure the artificial enzyme?

Answer 8

The artificial enzyme will have a colour, and you can directly measure the concentration of any molecule with a colour.

Question 9

What can some natural substrates show after conversion to product (think NADH and NAD+)

Answer 9

Some substrates can show absorbing changes

Question 10

How does NADH show an absorbance change?

Answer 10

Before getting oxidized, NADH absorbs light at the 340nM wavelength. Once oxidized to NAD+, it does not absorb light at that wavelength which can be used as a progress tracker and a way of measuring rate of reaction

Question 11

What happens to absorbance as substrate is converted into product?

Answer 11

Absorbance decreases.

Question 12

What are chromophores?

Answer 12

Conjugated double bonds

Question 13

What is a conjugated double bond?

Answer 13

The double bond is on every other bond (think aromatics)

Question 14

What is the wavelength of light a human can visibly see?

Answer 14

400-700 nM

Question 15

What wavelength can coloured compounds absorb?

Answer 15

400 - 700nM

Question 16

What wavelength of light can naturally biochemical chromophores absorb?

Answer 16

Between 200 - 400nM (much shorter)

Question 17

What type of enzyme is NADH

Answer 17

An oxidoreductase, also a chromophore

Question 18

What wavelength do larger chromophores absorb at?

Answer 18

Longer wavelength

Question 19

What machine measures absorbance?

Answer 19

A spectrophotometer (also known as a microplane reader), which will give you absorbance reading.

Question 20

What units does absorbance have?

Answer 20

Unitless, its a ratio: log(Io/l) therefore the units cancel out

Question 21

What does the Beer-Lambert Law Suggest?

Answer 21

That absorbance is proportional to sample concentration and sample thickness

Question 22

What is the Beer-Lamber Law?

Answer 22

A = e x c x l

e = molar extinction coefficent (mol l cm)
c = sample concentration (mol L)
l = sample thickness (cm)

*Absorbance is always unites

Question 23

What is the Rate of Reaction defined as?

Answer 23

The change in [substrate] or [product] per unit time
- Measured in mol/L/min

Question 24

What is the formula for Rate of reaction 1.)

Answer 24

1.) [Concentration] of Substrate Used/Time

2.) [Concentration] of Product Formed/Time

Question 25

What is Enzyme Activity measuring?

Answer 25

The amount of enzyme present

Question 26

What is the formula for Enzyme Activity?

Answer 26

Moles of Substrate/Time or Moles of Product/Time

*Can also be demonstrated by rate x volume

Question 27

What are the units of enzyme activity

Answer 27

umol/minute (1 enzyme unit)

Question 28

What is Specific Activity?

Answer 28

Measuring the purity of the enzyme during purification.

Question 29

What is the Specific Activity measuring

Answer 29

The number of enzyme per milligram of total protein.

Question 30

Formula for Specific Activity?

Answer 30

Enzyme Activity (umol/min)/Crude Protein(mg)

Question 31

What is the formula for Molar Activity?

Answer 31

The Specific Activity x Molar Mass of Enzyme

Question 32

What is Molar activity equal to?

Answer 32

The “turnover number” - The Number of Catalytic reactions per enzyme per second.

Question 33

What is a progress curve?

Answer 33

A post of substrate or product concentration over a period of time

Question 34

Where would the initial velocity be taken from?

Answer 34

The slope of the curve at time 0

Question 35

When you measure substrate concentration what is the slope?

Answer 35

-slope

Question 36

When you measure product concentration what is the slope

Answer 36

+slope

Question 37

What is your initial rate (Vo) equal to?

Answer 37

k2[ES]

Question 38

What does putting the initial rate of a function of time = 0 demonstrate?

Answer 38

The elimination of the reverse reaction.

Question 39

What is Vmax?

Answer 39

Vmax is the theoretical maximum velocity for enzyme concentration`

Question 40

What is Km

Answer 40

The Michaelis constant. Half of the maximum velocity given for substrate concentration

Question 41

What is Vmax?

Answer 41

The “upper limit”, where substrate concentration can increase but your rate can not increase (a plateau)

Question 42

What does Vmax indicate?

Answer 42

The catalytic rate: where 100% of the enzyme is occupied by the substrate.

Question 43

What does a higher Vmax mean

Answer 43

A faster reaction, better catalysis

Question 44

Why is Vmax considered a “pseudo” constant?

Answer 44

It is only a constant if the amount of enzyme is fixed
- if enzyme concentration increases, the Vmax will increase.

Question 45

What is K2?

Answer 45

K2 is a true constant, and is the turnover number (molar activity) of the enzyme