Regulation - keeping the system in balance

Question 1

Describe cell factory regulation

Answer 1

• regulation by mass action
• feedback loops & allosteric regulation
• regulation by spatial organisation of enzymes
• regulation by post-translational modification of enzymes

Question 2

Describe regulation by gene expression

Answer 2

changing the amounts of enzymes present

Question 3

Give an example of regulation by gene expression

Answer 3

• lacZ encodes beta-galactosidase
• lacY encodes beta-galactoside permease
• lacA encodes LacA, an
  enzyme that transfers an acetyl group from acetyl- CoA to β-galactosides

Question 4

Describe beta-galactosidase permeate

Answer 4

transmembrane transporter that pumps lactose into the cell

Question 5

LacA

Answer 5

β-galactoside transacetylase

Question 6

Describe feedback regulation of ATCase

Answer 6

• rate of the reaction catalysed by ATCase is fast at low concentrations of CTP
• slows as CTP concentration increases
• ATCase is inhibited by CTP

Question 7

ATCase

Answer 7

aspartate transcarbamoylase

Question 8

Describe the Carbamoyl phosphate pathway

Answer 8

• Carbamoyl phosphate + aspartate -> N-Carbomoylaspartate via ATCase
• • Pi -> CTP

Question 9

CTP

Answer 9

cytidine triphosphate

Question 10

Describe ATCase kinetics

Answer 10

• not Michaelis-Menten
• sigmoidal
• very large change in activity within a narrow substrate concentration range

Question 11

Describe ATCase

Answer 11

• consists of separate C and R subunits
• 6 catalytic subunits arranged as a pair of trimers, one sitting on top of the other
• 6 regulatory subunits arranged as three dimers that sit between the catalytic subunit junctions
• CTP binds to a site on each regulatory subunit

Question 12

C

Answer 12

catalytic

Question 13

R

Answer 13

regulatory

Question 14

Describe ATCase active sites

Answer 14

• each catalytic trimer has three active sites at the junction between catalytic subunits
• contains key amino acid residues from each of the two catalytic subunits that make it up

Question 15

Substrate binding and inhibitor binding

Answer 15

favour different conformational states

Question 16

Describe the R state

Answer 16

• catalytic subunit rotation brings active site residues closer together: more efficient catalysis
• catalytic triads are 1.2 nm further apart, favours substrate binding
• more active
• favoured by substrate binding

Question 17

Describe the T state

Answer 17

• less active
• favoured by CTP binding

Question 18

Describe the sigmoidal kinetics of ACTase

Answer 18

result of the combined kinetics of the T and R states of the enzyme

Question 19

Describe allosteric activation & inhibition of a single enzyme

Answer 19

solves the energy vs carbon skeleton partitioning problem in glycolysis

Question 20

Metabolic pathways typically contain

Answer 20

multiple feedback and feedforward loops to provide fine regulation of pathway flux in response to varying demands from the rest of the metabolic system

Question 21

Describe the biosynthesis IMP

Answer 21

• 10 steps
• catalysed by 6 enzymes
• organisation of enzymes into a cluster accelerates pathway flux

Question 22

IMP

Answer 22

• inosine monophosphate
• precursor for the synthesis of purines

Question 23

purines

Answer 23

adenine & guanine

Question 24

Describe enzyme clustering

Answer 24

• faster because of increased local concentration of enzyme
• prevents other pathways from competing for intermediates

Question 25

Why is phosphorylation an effective means of regulation?

Answer 25

• phosphoryl group adds two negative charges to the protein and can form 3 or more hydrogen bonds
• can take place in less than a second or over a span of hours
• produces highly amplified effects. One kinase can phosphorylate hundreds of target proteins in a short interval
• uses ATP as the phosphoryl donor and thus it is linked to the cell’s energy status

Question 26

Describe the addition of a phosphoryl group

Answer 26

alters the structural conformation of the enzyme and affect substrate binding and catalytic activity

Question 27

Describe phosphorylation and dephosphorylation

Answer 27

controlled by kinase and phosphatase enzymes