Sept 13 Protein Function and Regulation

Question 1

what activity is the basis of the many diverse functions of proteins?

Answer 1

binding
proteins bind to one another, macromolecules, small molecules and ions

Question 2

what is the molecule to which a protein binds?

Answer 2

the ligand

Question 3

what happens in some cases when the protein binds to its ligand?

Answer 3

ligand binding can change the conformation of the protein
ligand induced conformational change is integral to the mechanism of action of many proteins

Question 4

what are the two most important properties in ligand binding?

Answer 4

specificity and affinity

Question 5

what is specificity?

Answer 5

the ability of a protein to bind to only one particular ligand, even in the presence of a big excess of irrelevant molecules

Question 6

what is affinity?

Answer 6

the tightness/strength of binding, expressed as a dissociation constant Kd
stronger interaction –> lower Kd

Question 7

what does protein binding specificity arise from?

Answer 7

arises from numerous interactions which are individually weak but collectively strong
binding is an interaction between complementary molecular surfaces

Question 8

how do antibodies bind to their antigens and what allows them to do so?

Answer 8

antibodies bind to their antigens with high specificity and high affinity
antibodies two identical heavy chains and two identical light chains linked by disulfide bonds
antigen binding surface or CDR (complementarity determining region) involves multiple protein loops from both the heavy and light chains

Question 9

what are enzymes and what is their ligand called?

Answer 9

extremely diverse class of catalylitcally active proteins whose ligands are the substrates of the reactions they catalyse

Question 10

where does substrate binding and reaction catalysis occur?

Answer 10

occurs at the enzymes active’s site
the active site includes the substrate binding site (complementary to the specific substrate)
and the catalytic site which carries out the chemical reaction once the substrate is bound

Question 11

what is Vmax?

Answer 11

the maximal rate of catalysis given saturating amounts of substrate
depends on the amount of enzyme and how fast it can work
turnover number–> number of enzymatic cycles per second at top speedw

Question 12

what is Km?

Answer 12

the substrate concentration that supports a rate of catalysis equal to one half of the V max
depends on, and is a measure of the affinity of enzyme:substrate binding

Question 13

how do the values of Vmax and Km differ when you the concentration of enzymes changes?

Answer 13

the Vmax is higher, proportional to how many more enzymes were added
however the Km stays the same, because in general, binding affinity is independent of concentrations, but depends only on the chemical properties of the enzyme and the substrate?

Question 14

what does the fact that enzymes exhibit pH optima reflect?

Answer 14

1. evidence of active site acid base chemistry
2. sensitivity of the overall protein to charge distribution

Question 15

what happens to enzymes in a common pathway? (what is metabolic coupling)

Answer 15

there are three ways
1. the enzymes can become associated together into a quaternary structure which is much more efficient
each enzyme is coded for by a different gene
2. the enzymes can bind to a common “scaffold” protein
3. by evolution, enzymes can combine into a single polypeptide, coded for by a single gene
a multifunctional enzyme which has different domains(!!!)

Question 16

what is allosteric effect?

Answer 16

binding of a ligand at one site of a protein can lead to the conformational change that affect the binding of another ligand at a different site
can be used to control protein activity, turn it “on” or “off”

Question 17

in what other ways can protein conformation and activity be modified?

Answer 17

through phosphorylation and dephosphorylation
phosphorylation of amino acid side chains is a rapidly reversible covalent modification of protein structure –> post translational modification

Question 18

what needs to happen for high affinity and highly specific interactions to take place?

Answer 18

the shape and chemical properties of the binding site must be complementary to those of the molecule

Question 19

what is the induced fit model?

Answer 19

substrate binding site is not rigid, but flexible
and that substrate binding induces the enzyme to change shape and consequently bind more strongly to the transition state

Question 20

what is the enzyme substrate complex?

Answer 20

the bound substrate to the enzyme
the ES complex is in equilibrium with the unbound enzyme and substrate and is an intermediate step, followed by the conversion of the substrate to the product

Question 21

how do enzymes catalyse the process of a reaction?

Answer 21

they divide the it into multiple, discrete chemical reactions, in which the product of one reaction is the substrate for another
different ES complexes are generated before the final product

Question 22

what is a cofactor/prosthetic group?

Answer 22

helpergroup, non polypeptide small molecule or ion that is bound in the active site and plays an essential role in the reaction mechanism
also called coenzymes
some (not all) are chemically modified during the reaction and therefore need to be replaced or regenerated

Question 23

what are enzyme inhibitors?

Answer 23

bind to active sites and disrupt catalytic reactions

Question 24

what are competitive inhibitors?

Answer 24

bind directly on an enzyme’s binding site and this compete directly with the normal substrate

Question 25

what are non competitive inhibitors?

Answer 25

interfere with enzyme activity, in other ways, binding somewhere that is not the substrate binding site and change the enzyme’s conformation

Question 26

what does negative allostery often involve? and what does it do?

Answer 26

involves the end product of a multistep biochemical pathway
that end product will bind to the allosteric site of a enzyme that catalyses an early, rate controlling step in that pathway, therefore reducing its activity
excessive buildup of the product is prevented
it is called end-product inhibition

Question 27

what is cooperativity and its effects?

Answer 27

the influence (+ or -) that the binding of a ligand at one site has on the binding of a molecule of the same type of ligand on a differ site

Question 28

what is an example of cooperativity?

Answer 28

hemoglobin is a classic example of positive cooperativity
the binding of a single ligand (oxygen) increases the affinity of hemoglobin for the next oxygen molecule
(binding of one ligand increases sensitivity for another ligand)
cooperativity permits hemoglobin to take up oxygen very efficiently in the lungs and unload in tissues
provides a selective evolutionary advantage

Question 29

what is phosphorylation?

Answer 29

reversible addition of a phosphate group from ATP to a hydroxyl group of a side chain of a serine threonine or a tyrosine residue
changes the protein activity

Question 30

what enzymes are involved in phosphorylation and dephosphorylation?

Answer 30

phosphorylation is catalysed by kinases
dephosphorylation is catalysed by phosphatases

Question 31

what can phosphorylation and dephosphorylation influence? (5)

Answer 31

1. location of a protein within cells
2. structure and intrinsic activity
3. its ability to bind to other molecules
4. its ability to undergo further covalent modifications
5. its stability

Question 32

which proteins are regulated by kinase and phosphatase modifications?

Answer 32

all classes of proteins

Question 33

how is a cascade effect created by phosphorylation and dephosphorylation?

Answer 33

the target of a kinase or phosphatase is yet another kinase or phosphatase

Question 34

what is the definition of binding?

Answer 34

an interaction between complementary molecular surfaces

Question 35

explain the mechanism of serine protease trypsin as an example of enzyme-substrate binding and catalytic site specificity:

Answer 35

proteases hydrolyse peptide bonds in polypeptides
the serine proteases are a family of proteases whose catalytic mechanism involved a serine residue in the catalytic site
trypsin hydrolyses peptide bonds (digestion) adjacent to arginine and lysine, which are large basic side chains
proper substrate binding only occurs when the substrate amino acid side chain “fits” into the negatively charged pocket within the substrate binding side
the different substrate recognition pockets in enzymes define their specificity
positive arginine interacts with the negative side chain from the enzyme
arginine stays in position long enough for catalysis to happen

Question 36

why do proteins fold?

Answer 36

to bring distant amino acids into proximity, in a way that is chemically effective

Question 37

explain the mechanism of trypsin in detail (see slide 9 of lecture)

Answer 37

Step 1: cleavage of a peptide bond with formation of a covalent substrate enzyme complex
a. oxygen from Serine side chain attacks the carbonyl carbon on the polypeptide backbone
this is facilitated by the fact that histadine is basic, it can readily accept protons and obtain a positive charge
b. the oxygen and carbon are now covalently bonded, the carbon is now part of the enzyme (transition state)
electrons that form the peptide bond pull back hydrogen proton from histadine
c. by condensation, fragment can now break away, while part with N terminal is still attached

step 2: hydrolysis of acyl enzyme complex
d. with water, the bond between the oxygen (from the enzyme) and the carbonyl carbon is hydrolysed
the electrons on the oxygen from water attack the carbonyl carbon
again, this is facilitated by histadine
e. a proton from water is picked up by histadine
f. the bond between the oxygen and the carbonyl carbon is broken, and the protein diffuses away
the active site is regenerated like at the beginning

Question 38

what are two allosteric switches that are widely used to control protein activity?

Answer 38

noncovalent binding of Ca2+ and GTP

Question 39

what is the effect of Ca2+ binding to calmodulin?

Answer 39

it changes its conformation, allowing it to bind to target peptides on other proteins, thus regulating their structure and activity
calmodulin can turn on other proteins
Ca2+ activates calmodulin, which activates other proteins

Question 40

what are the two settings of G-proteins and what is their effect?

Answer 40

“on” (GTP bound) and “off” (GDP bound) conformations that interact differently with other proteins
switching from on to off is facilitated by GAPs and switching from off to on is facilitated by GEFs