Lecture 23 - Protein function and regulation Flashcards

1
Q

Common activity of all proteins

A

Binding

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2
Q

Name of what proteins bind to and consequence of binding

A

Bind to ligand -> Conformational change that may result = the protein does its function

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3
Q

2 important properties in ligand-binding

A

Specificity and Affinity

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4
Q

What do we mean by the specificity of a protein

A

Its abiliy to bind a particular ligand even when in presence of many irrelevant molecules

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5
Q

What do we mean by the affinity of a protein and how is measured

A

Strength of binding. Dissociation constand (Kd). Lower Kd = stronger binding

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6
Q

What makes binding possible (how is it possible if bonds between molecules are very weak)

A

Summation of all interactions between 2 protein surfaces makes binding

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7
Q

4 things that are good for binding of 2 surfaces

A

1) Surfaces have complementary shapes
2) H bonds
3) Complementary charges (plus one side, minus other)
4) Hydrophobic interactions

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8
Q

Best molecules for specificity and affinity + how they bind

A

Antibodies. Bind with CDR (complementarity determining region)

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9
Q

Where CDR found on antibody/what it’s made of

A

On both ends of the Y, made of loops of heavy chain and light chain that are highly variable

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10
Q

Enzymes : Special thing about their ligands

A

Are the substrates of the reactions they catalyze

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11
Q

General scheme of reaction from energy POV

A

Reactants energy, Transition state (activation energy). Products energy

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12
Q

What enzymes change in a reaction (2)

A

1) Reduce activation energy

2) Therefore, increase reaction rate

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13
Q

where on enzyme do substrate (ligand) binding and reaction catalysis occur 2 components of this region

A

In enzyme’s active site. Has a substrate binding site and a catalytic site

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14
Q

How rigid is the binding of an enzyme to its substrates + name of phenomenon by which they fit together

A

Fluid. There is some molecular flexibility which allows an induced fit of substrate in its binding site

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15
Q

What makes up an enzyme’s catalytic site and substrate binding site

A

Amino acids that are close by on its surface (3D shape) but that could be far in the linear polypeptide

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16
Q

What are proteases

A

Proteases cleave proteins (hydrolyze peptide bonds).

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17
Q

what kind of protease trypsin is + why

A

Trypsin = serine protease cause catal. mechanism involves key serine (serine 195) w/ OH group

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18
Q

Specific activity of trypsin (where it cuts)

A

Cuts between lysine and arginine residues

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19
Q

Special name of substrate binding site in trypsin

A

Side-chain-specificity binding pocket

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20
Q

What goes in the side-chain-specificity binding pocket and how it’s held there

A

Arg or Lys positively charged side chain. Held by negatively charged aspartate

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21
Q

Beside interaction on the substrate binding site, how can interactions also occur between substrate and enzyme

A

Possible interactions between enzyme and substrate backbones (H bonds) elsewhere

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22
Q

What determines specificity of the enzyme

A

ONLY the interaction between peptide and substrate binding site (aspartate and side chain of residue at Arg position)

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23
Q

2 other serine proteases like trypsin but with different specificities

A

Chymotrypsin and elastase

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24
Q

Chymotrypsin substrate binding site (side-chain-specificity binding pocket)

A

Contains a serine so less specificity than trypsin for basic residues

25
Q

Elastase substrate binding site (side-chain-specificity binding pocket)

A

Contains bulky amino acids like valine so cleaves beside a.a s with small side chains

26
Q

3 key amino acids in the catalytic mechanism of trypsin and other serine proteases

A

Ser 195, his 57 and asp 102

27
Q

First step of serine proteases catalytic mechanism

A

Cleavage of peptide bond to free P2 N-termini and formation of an acyl complex

28
Q

Second step of serine proteases catalytic mechanism

A

Hydrolysis of acyl enzyme complex

29
Q

Intermediate in first and second step of serine proteases catalytic mechanism + special name and why

A

Tetrahedral intermediate called an oxyanion cause it has a single bond to an oxygen that is negatively charged

30
Q

First general rule of enzyme catalysis

A

Enzyme catalysis based on stabilized binding of transition state

31
Q

Second general rule of enzyme catalysis

A

Enzyme catalysis based on 3D organization of key amino acids in the active site

32
Q

First common observation/pt of interest about enzymes

A

May break down a reaction into several sub-reactions

33
Q

Second common observation/pt of interest about enzymes

A

Enzyme’s active site chemistry may be pH dependent

34
Q

Why pH influences enzyme activity (so why they have an optimal pH)

A

1) pH influences active site acid-base chemistry

2) pH influences conformation of whole protein

35
Q

Exemple of enzymes that adapted to work at low pH and how they did it

A

lysosomal hydrolases -> use different catalytic mechanism

36
Q

Why chymotrypsin doesn’t work at pH lower than 8

A

His-57 becomes already protonated so can’t steal proton (1st step in mech.)

37
Q

What Michealis-Menten enzyme kinetics show

A

Rate of an enzymatic reaction can be expressed as function of substrate concentration

38
Q

What is the Vmax

A

Maximal rate of catalysis given saturating amounts of substrate

39
Q

What influences Vmax

A

Amount of enzyme and turnover number

40
Q

What is the turnover number

A

maximum number of substrate molecules converted to product per enzyme molecule per second.

41
Q

V max formula

A

Enzyme (site) concentration * turnover number

42
Q

What is Km

A

substrate concentration that supports a rate of catalysis equal to half the Vmax

43
Q

Does the enzyme qt influence the Km

A

No. No matter the Vmax (influenced by enzyme qt) Km is always the same for a given enzyme-substrate pair

44
Q

Why Km is constant for an enzyme-substrate pair

A

Binding affinity independent of concentrations and depends only on chemical properties of enzyme + substrate

45
Q

Ways to accelerate effect of enzymes involved in a common pathway (of reactions) (3)

A

Binding of enzymes together
Binding of enzymes on a common scaffold
Link enzymes’ genes to make a common multienzyme complex

46
Q

Name of binding of many enzymes together and utility

A

Multienzyme complexes. Products from a catalyzed reaction are easily available for next one

47
Q

Name of enzymes’ genes linked into one enzyme and characteristic

A

Multifunctional enzyme. Many different domains and catalytic sites with related reactions

48
Q

What are allosteric effects

A

Change of protein conformation at a site other than ligand binding site

49
Q

Consequence of allosteric effects

A

Protein may be able to accomplish new function

50
Q

2 major manifestations

A

1) Cooperative binding of substrates to multimeric protein complexes
2) Conformational switches in regulatory proteins in response to post-transl. modif (or ligand binding obviously)

51
Q

Example of cooperative binding in the blood

A

When one O2 molecule binds hemoglobin, increases Hb affinity to other O2 molecules and they will bind more readily (conformational change)

52
Q

Shape of O2 binding curve and what each axis represents

A

Sigmoidal shape (S-shape) on graph pO2 on X and Saturation of Hb w/ O2 on Y axis

53
Q

2 examples of allosteric switches

A

Noncovalent binding of Ca 2+ and GTP

54
Q

Example of allosteric switch w/ calcium binding

A

When calmodulin binds calcium, it can adopt proper shape to bind target peptides (thus regulating their structure/activity)

55
Q

Explanation of allosteric switch w/ GTP

A

Proteins that are GTPases regulated by GTP and can hydrolize it
GTP -> GDP : becomes inactive and step accelerated by GAP (GTPase activating protein)
GDP -> GTP : becomes active and step accelerated by GEF (guanine nucleotide exchange factor)

56
Q

On what structure of a protein phosphorylation/dephosph. of a protein is done and bond nature

A

Phosphorylation of amino acid SIDE CHAINS -> COVALENT bond

57
Q

How phosphorylation/dephosph. can affect protein activity

A

Phosphorylating protein can activate it and dephosphorylating it can inactivate it

58
Q

What proteins regulate phosphorylation /dephosphorylation and how

A

Protein kinases phosphorylate (add a Pi to protein and an ATP becomes ADP), protein phosphatases dephosphorylate (remove Pi w/ water)

59
Q

How many protein kinases we have

A

More than 500 diff. kinases in human genome