Enzymes

Question 1

What is K_cat?

Answer 1

K_cat is equivalent to the number of substrate molecules a single molecule of enzyme can bind and convert to product every second (when substrate is unlimited)

Question 2

Enzyme activity can be measured by determining one of which 3 things?

Answer 2

• rate of production of product
• rate of loss of substrate
• rate of production of loss or cofactor involved in the reaction

Question 3

What is an enzyme activity unit?

Answer 3

The amount of enzyme which transforms 1 micromole of substrate per minute at 25°^C

Question 4

What is the specific activity of an enzyme?

Answer 4

The number of enzyme units per milligram of protein

Question 5

What is the function of triose phosphate isomerase?

Answer 5

Catalyses the interconversion of DHAP and G3P in glycolysis

Question 6

What are the units of K_m?

Answer 6

Same as the units of substrate concentration

Question 7

What is on the y axis of a Lineweaver-Burk plot?

Answer 7

1/V (rate of reaction)

Question 8

What is on the x axis of a Lineweaver-Burk plot?

Answer 8

1/[Substrate]

Question 9

What is the y intercept on a Lineweaver-Burk plot?

Answer 9

1/V_max

Question 10

What does the x intercept on a Lineweaver-Burk plot represent?

Answer 10

-1/K_m

Question 11

What is V_max?

Answer 11

Rate of reaction when an enzyme is saturated with substrate

Question 12

What is K_m?

Answer 12

The concentration of substrate which permits the enzyme to achieve half V_max

Question 13

Why is enzyme inhibition important biologically?

Answer 13

Acts as a mechanism for controlling enzyme catalysed reactions in vivo

Question 14

How do drugs inhibit HIV protease?

Answer 14

They mimick the natural peptide substrate and blocks the active site

Question 15

Does prescence of a competitive inhibitor affect K_m?

Answer 15

Yes, it increases it

Question 16

Does prescence of a competitive inhibitor affect V_max?

Answer 16

No

Question 17

Does prescence of a non-competitive inhibitor affect Vmax?

Answer 17

Yes, it reduces it

Question 18

Does prescence of a non-competitive inhibitor affect K_m?

Answer 18

No

Question 19

Give an example of an inhibitor used to regulate enzyme activity

Answer 19

Pancreatic trypsin inhibitor

Question 20

What is the structure of COX enzymes?

Answer 20

Dimer with haem group at active site

Question 21

How can competitive and non-competitive inhibitors be easily distinguished kinetically?

Answer 21

• Compare the Lineweaver-Burk plot for reaction in prescence and abscence of inhibitor
  *

Question 22

Outline 6 main contributions to the catalytic activity of enzymes

Answer 22

• Enhanced reactivityof side chains in amino acids in active site
• Proximity and orientation effects
• Acid-base catalysis
• Covalent catalysis
• Stabilisation of transition state
• Utilisation of metal ions in catalysis

Question 23

How is proximity and orientation important in the catalytic activity of enzymes?

Answer 23

• binding of substrate orients it with respect to catalytic groups of enzyme (esp. important for stereospecificity)
• electrostatic steering of substrate towards active site

Question 24

What is the function of L-lactate dehydrogenase?

Answer 24

Stereospecific conversion of pyruvate to L-lactate under anaerobic conditions

Question 25

What is the structure of L-lactate dehydrogenase?

Answer 25

Tetrameric enzyme

Question 26

What cofactor is required by L-lactate dehydrogenase?

Answer 26

NADH

Question 27

Outline the mechanism of L-lactate dehydrogenase

Answer 27

* Ternary complex formed * Hydride transfer from NADH * Protonation of pyruvate C=O by His * Stereospecific L-lactate produced * Active site loop closure

Question 28

How does the stereospecificty of L-lactate dehydrogenase arise?

Answer 28

Pyruvate binds in the active site in a very specific orientation due to groups in side chain of active site

Question 29

Which 3 amino acids are involved in the catalytic triad in serine proteases?

Answer 29

* Serine * Histidine * Aspartate

Question 30

What kind of amino acids can the chymotrypsin specificity pocket accomodate?

Answer 30

Large hydrophobic amino acids

Question 31

What kind of amino acids can the trypsin specificity pocket bind to?

Answer 31

Positively charged amino acids due to a negative charge in the pocket

Question 32

How does L-lactate hydrogenase have stereospecificity?

Answer 32

Pyruvate is held in the active site in such an orientation hydride can only enter from one side

Question 33

Name an example of a serine protease

Answer 33

Chymotrypsin

Question 34

What is the evidence for the mechanism of serine proteases?

Answer 34

* The rate of the reaction seems to occur in 2 phases which suggests a covalent intermediate is formed * Chymotrypsin is inactivated by DIPF which specifically reacts with ser195 whilst the rest of the serines do not, suggesting that ser195 has enhanced reactivity

Question 35

What is the function of the oxyanion hole?

Answer 35

Stabilises the negative charge on the oxygen as the NH groups point downwards and stablise the O^-

Question 36

How is chymotrypsin activated?

Answer 36

By proteolysis of trypsin

Question 37

How does trypsin activate chymotrypsin?

Answer 37

* In the preform of chymotrypsin the active site is strained and not fully formed * Trypsin cleaves a peptide bond which produces a working active site

Question 38

What activates all serine proteases?

Answer 38

Trypsin

Question 39

What activates trypsin?

Answer 39

Enteropeptidase

Question 40

What inactivates trypsin?

Answer 40

Pancreatic trypsin inhibitor

Question 41

Give an example of a serine protease which was formed due to convergent evolution

Answer 41

Carboxypeptidase II

Question 42

Outline how divergent evolution has led to different forms of serine proteases

Answer 42

* ancient gene coding for the catalytic triad is duplicated through a gene duplication process * there are now 2 genes coding for the same enzyme * this allows one to mutate whilst the other maintains function

Question 43

Why is it important to measure the initial rate of an enzyme catalysed reaction?

Answer 43

Rate of reaction begins to tail off, especially at high concentrations of enzyme