lecture 2 biochemistry (week 1)

Question 1

what are motifs?

Answer 1

conserved sequences of amino acids that can suggest functional characteristics

Question 2

what are domains?

Answer 2

domains are distinct, autonomous, and functionally independent units within a protein sequence

Question 3

where are protein kinases and phosphates found to be working?

Answer 3

normally as part of signalling cascades and usually amplify the signals

human genome encodes ~ 520 kinases & ~ 150 phosphatases. Ser/Thr kinases are most well studied.

His kinases also exist but rare.

good example of protein family - conservation evident (structure + mechanism)

Question 4

what has bioinformatics revealed about kinases?

Answer 4

revealed the extant of kinases in the genome (>500) help ID key catalytic residues can use sequence alignments to reveal evolutionary and regulatory similarities

Question 5

what is the adenyl cyclase pathway?

Answer 5

ligand binds –> adenyl cyclase hydrolyses ATP to cAMP –> cAMP activates PKA –> cellular response

Question 6

what is cAMP?

Answer 6

important intracellular second messenger

Question 7

describe the R2C2 complex

Answer 7

2 regulatory and 2 catalytic subunits –> cAMP binds R subunit –> activates C subunit

Question 8

describe the mechanism of ATP binding to protein kinases

Answer 8

ATP binds the active site of the kinase –> substrate binds the active site - y-phosphate is transferred from ATP to a Ser/Thr/Tyr –> substrate released –> ADP released

Question 9

what is a pseudosubstrate?

Answer 9

small sequence of amino acids that mimics the substrate and binds substrate binding cavity in the catalytic domain; lacks Ser/Thr acceptor residues = enzyme inactive

interaction with membrane –> release of PS –> activation of enzyme (DAG binds C1) only happens if Ca + DAG

Question 10

what are the conserved sequence motifs?

Answer 10

glycine rich pool = GGG

beta3 Lys/Arg = K (RC)

aC helix Glu = E

catalytic loop Asp/Asn = DN

DFG motif Asp = D

Question 11

describe the target specificity of the target proteins

Answer 11

sequences around the phospho-acceptor site show similarity allowing the prediction of other sites in other proteins using bioinformatics

Question 12

describe the changes phosphorylation can induce

Answer 12

structure - change conformation and ability/selectivity of interactions

change localisation

change interactions

change half life

change sensitivity to signals

Question 13

what is the activation loop?

Answer 13

physically blocks access to the active site - keeps kinase inactive

Question 14

describe the displacement of the activation loop.

Answer 14

the activation is phosphorylated which moves the loop ‘out of the way’ of the active site

Question 15

what are the phosphorylation events called involving the activation loop?

Answer 15

priming events

Question 16

what is the PKC superfamily made up of?

Answer 16

domains

Question 17

what is the pseudosubstrate domain?

Answer 17

small sequence of amino acids

mimics substrates

bind substrate binding cavity in the catalytic domain

lacks serine or threonine phospho-acceptor residues therefore the enzyme stays inactive

Question 18

describe the process of the pseudosubstrate binding

Answer 18

interaction with the membrane –> release of pseudosubstrate –> activation of the enzyme

Question 19

how is the binding of the pseudosubstrate controlled?

Answer 19

DAG bids to C1 and this can only happen when Ca is present

Question 20

how is bioinformatics used to predict

Question 21

what can phosphorylation cause?

Answer 21

change localisation of proteins

change interactions (increase/decrease affinity)

mediate selective interactions

change half life

change sensitivity to a signal