regulation of protein function

Question 1

why are proteins regulated?

Answer 1

to ensure:
maintenance of cellular homeostasis
responsiveness to the environment
efficiency

Question 2

what are the major ways in which enzyme activity can be regulated?

Answer 2

• changes in substrate concentration
• binding of small effector molecules
• reversible covalent modifications
• binding of regulatory proteins
• proteolytic activation
• controlling the amount of enzyme present

Question 3

how can controlling substrate concentration regulate protein function?

Answer 3

• affects rate

- particularly important when [S] is less than Km, when rate is linearly dependent on [S]

Question 4

what are isoenzymes?

Answer 4

enzymes that catalyse the same reaction but have different amino acid sequences

Question 5

what are the roles of isoenzymes?

Answer 5

• can have different metabolic uses in different organs
• can have different locations and metabolic roles in the same cell
• can have different roles at stages of development
• can have different responses to allosteric regulators

Question 6

give an example of an isoenzyme

Answer 6

lactate dehydrogenase

• main types: H4 + M4
• H4 is found mainly in the heart, has low Km for lactate and favours lactate oxidation
• M4 is found in skeletal muscle, has low Km for pyruvate and favours pyruvate reduction

Question 7

what is allosteric regulation?

Answer 7

regulation mediated by interactions of a modulator at a regulatory site away from the active or binding site. binding of a modulator causes a conformational change

Question 8

what is a homotropic modulator?

Answer 8

an allosteric modulator which is the same as the substrate

Question 9

what is a heterotropic modulator?

Answer 9

an allosteric modulator which is different from the substrate

Question 10

do allosterically modulated enzymes obey Michaelis-Menten kinetics?

Answer 10

no

Question 11

give an example of allosteric regulation

Answer 11

co-operative oxygen binding in haemoglobin

haemoglobin undergoes a structural change on binding Hb, oxygen binding promotes stabilisation of high affinity R state

Question 12

what do allosteric activators do?

Answer 12

increase the proportion of enzyme in the R state

Question 13

what do allosteric inhibitors do?

Answer 13

increase the proportion of enzyme in the T state

Question 14

when are covalent modifications made?

Answer 14

post-translationally

Question 15

how do protein kinases cause covalent modification?

Answer 15

protein kinases transfer their terminal phosphate from ATP to the OH group of Ser/Tyr/Thre

Question 16

how do protein phosphatases cause covalent modification?

Answer 16

catalyse the hydrolytic removal of phosphoryl groups from proteins

Question 17

why is phosphorylation an effective covalent modification for regulation of protein?

Answer 17

• large free energy permits changes in conformation of phosphorylated protein
• addition of 2 negative charges disrupts existing electrostatic interactions
• phosphoryl group can form 3 new H bonds, allowing specific interactions with hydrogen bond donors
• permits amplification cascades

Question 18

explain how phosphorylation by kinases results in signal amplification?

Answer 18

activation of enzymes by other enzymes increases number of affected molecules - allowing signal amplification of several orders of magnitude in a few ms

Question 19

how is PKA an example of phosphorylation regulation?

Answer 19

in the absence of cAMP a pseudosubstrate region blocks activity of PKA
PKA consists of regulatory subunit and catalytic subunit
when cAMP binds to R subunit, it dissociates, releasing the C subunit which is catalytically active

Question 20

what is the concerted model for allostery?

Answer 20

one substrate molecule causes complete change in R state

Question 21

what is the sequential model for allostery?

Answer 21

substrate binding to one subunit will only affect adjacent subunits

Question 22

why is CTP an ATCase inhibitor?

Answer 22

CTP favours the R state, stabilising the inactive state

Question 23

what is the difference in response for allosteric enzymes vs those which obey Michaelis-Menten kinetics?

Answer 23

allosteric enzymes can respond to small changes in [S] compared to those which obey michaelis-menten

Question 24

how do adaptor proteins spatially restrict signals in the cell?

Answer 24

a-kinase anchoring proteins have different domains specific for PKA, PP2A, the target protein and specific regions of the cell membrane

Question 25

describe the structure of calmodulin

Answer 25

calcium is bound between two a-helices and a short loop, it binds via 7 oxygen atoms. there are 4 EF hand motifs to provide 4 Ca2+ binding sites.

Question 26

how is calmodulin regulated by calcium?

Answer 26

calcium binding to calmodulin causes a conformational change in the protein, and binds exposed a-helices on the target protein

Question 27

how does calcium regulate protein phosphatase calcineurin?

Answer 27

in the inactive state, an auto inhibitory domain blocks the active site, calcium binding triggers a change in conformation that unblocks the active site

Question 28

how can proteins be regulated by partial proteolysis?

Answer 28

precursors (pro proteins or zymogens) can be synthesised and removed in a highly specific, irreversible process to release the active protein

Question 29

how are active zymogens switched off?

Answer 29

tight binding of inhibitors or degradation

Question 30

how can the amount of protein present in the cell be regulated in the long term?

Answer 30

change in the rate of protein synthesis, enzyme repression

Question 31

how does the ubiquitin-proteasome pathway affect the regulation of the amount of protein in the cell?

Answer 31

changes the rate of protein degradation

Question 32

how can the catalytic activity of enzymes be controlled?

Answer 32

feedback regulation