11 - Enzymes VI: Activity and regulation

Question 1

Factors affecting enzyme activity

Answer 1

• Inhibitors
• Physical factors
• Cellular regulation

Question 2

Physical factors: temperature

Answer 2

• As temperature increases, collisions between substrates and active sites occur more frequently as molecules move faster
• Thermal agitation disrupt the weak bonds that stabilise the protein conformation, leading to thermal denaturation

Question 3

Physical factors: pH

Answer 3

• pH influences protein conformation and electrostatic interactions between enzyme and substrate (ionisation).
• Optimum between pH 6–8 for most enzymes
• However, digestive enzymes in the stomach are designed to work best at pH 2 while those in the intestine are optimal at pH 8, both matching their working environments

Question 4

How much enzyme activity is present in a cell?

Answer 4

A. Two major factors:
1. How much enzyme is present
– A balance between rates of synthesis & degradation
2. The absolute activity of the enzyme present
– Regulation

Question 5

changes in [S] and [P]

Answer 5

• [S] - more substrate means more product
• High [P] can inhibit enzyme activity - Product inhibition
BUT:
• [S] and [P] only affect overall measured rates of reaction and not the catalytic properties of an individual enzyme.

Question 6

Feedback inhibition

Answer 6

• Biochemical pathways often have a committed step:
- The first dedicated reaction (e.g. only final product is F)
- Often the rate-limiting step
• Final product of pathway often behaves as an inhibitor of the enzyme that catalyses the committed step.

Question 7

Feedback inhibition: 3-PGDH

Answer 7

3-phosphoglycerate dehydrogenase
Tetramer of 4 identical subunits:
• 4 catalytic sites
• 4 regulatory serine-binding sites - lowers Vmax for 3-PG
• Preserves 3-PG levels in the cell

Question 8

Allosteric regulation

Answer 8

• Usually associated with multi-subunit enzymes
• There may be positive and negative regulators involved
– Positive: cooperative binding
– Negative: allosteric inhibitors
• Allows feedback control of metabolic pathways
– Input and output

Question 9

Allosteric effect

Answer 9

an effect at one site on a molecule caused by binding of a molecule at a second, distinct site

Question 10

Positive cooperativity

Answer 10

• Allosteric enzymes do not obey Michaelis-Menten kinetics
• Rate vs. [S] plot is sigmoidal
• Substrate binding at one site decreases the KM at other active sites
• Results in rapid responses to increases in [S]i.e. steeper curve

Question 11

Sigmoidal plot

Answer 11

combination of two Michaelis-Menten type plots:
• T-state = “tense” - low activity
• R-state = “relaxed” - high activity
• Switch from T to R decreases KM & increases reaction rate

Question 12

Aspartate carbamoyltransferase (ACTase)

Answer 12

• ACTase is the first committed step in pyrimidine synthesis
• End points are nucleotides, including cytidine triphosphate
• Classic example of allosteric regulation
• Activity stimulated by the substrate aspartate -Positive allosteric regulation
• Activity inhibited by CTP - Negative allosteric regulation

Question 13

ACTase structure (multi-subunit)

Answer 13

• Catalytic subunit
– Binds substrates
– Catalyses formation of products
– Not affected by CTP
• Regulatory subunit
– Binds CTP
– No catalytic activity
Complete structure has 6 catalytic & 6 regulatory subunits

Question 14

ACTase structure (general)

Answer 14

Two layers of catalytic trimers surrounded by regulatory dimers

Question 15

ACTase: reaction mechanism

Answer 15

• PALA is a stable transition state analogue- forms a stable complex for structure determination

Question 16

ACTase: cooperative binding

Answer 16

• Substrate-binding induces a conformational change

Question 17

ACTase: allosteric inhibition

Answer 17

• CTP binds the regulatory subunits and stabilises the T-state

Question 18

ACTase: allosteric regulation

Answer 18

• Enzyme activity is regulated by the balance between substrate and end-point product.

Question 19

Isozymes/Isoenzymes

Answer 19

• Different versions of the same enzyme with different catalytic properties
– e.g. KM, Vmax, response to regulatory molecules.
• Encoded by different genes - gene families.
• Allows the same reaction:
– to have different affects on metabolism in different tissues,
or
– to take place under different conditions.
• Allows for fine tuning of metabolism.

Question 20

Lactate dehydrogenase (LDH)

Answer 20

Involved in both glucose metabolism and glucose biosynthesis

Question 21

Lactate dehydrogenase isozymes

Answer 21

LDH occurs as two isozymes:

• H form
• M form

Question 22

• H form ■ (Heart)

Answer 22

– higher affinity for substrates
– allosterically inhibited by pyruvate
– optimised to convert lactate to pyruvate to provide fuel for aerobic metabolism

Question 23

• M form ● (skeletal Muscle)

Answer 23

– faster catalysis

– optimised to convert pyruvate to lactate to allow anaerobic glycolysis

Question 24

Lactate dehydrogenase isoforms

Answer 24

M and H isozymes make different combinations of tetramer to give 5 major isoforms of LDH
• LDH-1 : 4 × H – primarily in heart muscle and red blood cells.
• LDH-2 : 3 × H, 1 × M – highest in white blood cells.
• LDH-3 : 2 × H, 2 × M – highest in the lung.
• LDH-4 : 1 × H, 3 × M – highest in the kidney, placenta, and pancreas.
• LDH-5 : 4 × M – highest in the liver and skeletal muscle

Question 25

LDH developmental regulation

Answer 25

• Isoform composition changes during rat heart development

- anaerobic (womb) to aerobic environment (breathing)