Enzyme regulation

Question 1

Enzyme regulation is

Answer 1

~ dynamic
~ complex
~ operates over multiple timescales

Question 2

Allosteric regulation

Answer 2

when binding of a small molecule/ligand induces a conformational change in the shape of the enzyme, AND the shape change brings about a change in the activity of that enzyme

Question 3

On/off transcriptional control

Answer 3

one possible strategy for regulating enzymes

Question 4

Which factors need to be taken under consideration when thinking about enzyme regulatory mechanisms?

Answer 4

~ enzyme half-lives
~ rate of transcription
~ rate of protein synthesis

Question 5

What types of regulation occurs over short timescale?

Answer 5

allosteric regulation

Question 6

What types of regulation occurs over moderate timescale?

Answer 6

~ covalent modification (e.g. phosphorylation)
~ compartmentalisation
~ aggregation (forming oligomers)

Question 7

What types of regulation occurs over longer timescale?

Answer 7

~ changes in gene expresson

~ hormonal regulation?

Question 8

Co-operativity

Answer 8

activity at one functional site affects activity at other functional sites

Question 9

ATCase

Answer 9

~ aspartate transcarbamoylase
~ E. coli
~ involved in pyrimidine biosynthesis
~ ATP stimulates it
~ CTP inhibits it (product produced further on down the line)

Question 10

ATCase structure (5)

Answer 10

~ regulatory dimer
~ r chain
~ catalytic trimer
~ c chain
~ zinc domain

Question 11

What are the two states that allosteric enzymes exist in?

Answer 11

T = tensed = low affinity
R = relaxed = high affinity = catalytic units are further apart + rotated

Question 12

What is the mechanistic effect of allosteric activation and inhibition?

Answer 12

1) ATPase does not follow Michaelis-Menton kinetics –> exhibits a sigmoidal curve
2) composite of two M-M curves –> one R, one T
3) CTP increases the initial phase of the s curve
4) ATP increases rate of reaction

Question 13

Monod’s concerted model

Answer 13

1. Allosteric enzymes exist in ONLY 2 states (T and R), and these states ARE IN EQUILIBRIUM
2. Upon binding ligand the enzyme stabilised in one of these confirmations (T or R)

Question 14

Koshland’s Sequential Model

Answer 14

1. conformational change in one subunit does not lead to change in ALL other subunits
2. conformation change of subunits is sequential –> one or some, not all
3. interactions between sub-units can be +ve or -ve

Question 15

What models are there to explain allosteric behaviour?

Answer 15

~ Concerted

~ Sequential

Question 16

Name the three different approaches for studying allosteric enzymes

Answer 16

~ x-ray crystallography
~ substrate analogues
~ site-directed mutagenesis

Question 17

Substrate analogues

Answer 17

chemical compounds with a chemical structure that resemble the substrate molecule in an enzyme-catalyzed chemical reaction
e.g. PALA

Question 18

PALA

Answer 18

~ substrate/transition state analogue

~ reaction intermediate and potent inhibitor

Question 19

X-ray crystallography + substrate analogues

Answer 19

~ allows us to view reaction intermediates

~ allows us to understand and visualise the reaction mechanism

Question 20

Enzymes have the highest affinity for…

Answer 20

transition state intermediates

Question 21

Name two other structural techniques. (excluding X-ray crystallography)

Answer 21

~ NMR
~ cryo-EM
~ provides structural characterisation at atomic resolution

Question 22

Site-directed mutagenesis

Answer 22

~ when mutation occurs in potential active site residues, it should kill the enzymes activity
~ possibility of enhancing/prevention cooperativity