KH5

Question 1

what is the activity that proteins must do to preform their diverse functions

Answer 1

BINDING
they bind to each other, other macromolecules, small molecules and ions

Question 2

what is a ligand

Answer 2

molecule that a protein binds to

Question 3

what happens when ligand binds sometimes

Answer 3

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

Question 4

name the 2 important properties in ligand binding

Answer 4

specificity
affinity

Question 5

what is specificity

Answer 5

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

Question 6

what is affinity

Answer 6

tightness or strength of binding
expressed as dissociation constant (Kd)
stronger interaction = lower Kd
weak interaction = high Kd

Question 7

what is protein binding

Answer 7

interaction between complementary molecular surfaces - like a puzzle piece
arises from numerous interactions - individually weak but collectively strong

Question 8

describe a stable complex vs less stable complex in protein binding

Answer 8

stable = puzzle piece fit, grooves match up, ionic bond, hydrogen bond, hydrophobic and van der waals interactions, charges attract
less stable = more irregular fit, no consistent pattern of complementarity, charges repel and disrupts

Question 9

describe antibody binding properties

Answer 9

bind antigens with high specificity and high affinity

Question 10

describe how antigens and antibodies bind

Answer 10

antibodies have 2 heavy chains and 2 light chains
antigen binding surface or CDR (complementarity determining region) involves multiple protein loops from both the hc and lc
loops are highly variable in aa sequence among antibody encoding genes - many possible cdrs = many possibilities

Question 11

what are enzymes

Answer 11

diverse class of catalytically active proteins
ligands include the substrate of the reactions they catalyze

Question 12

where do substrate binding and reaction catalysis happen

Answer 12

at enzymes active site

Question 13

what does substrate specificity arise from

Answer 13

substrate binding site
substrate and binding site interact at complementary molecular surfaces

Question 14

what do aas contribute to (substrates/enzymes)

Answer 14

some contribute to substrate binding site and others to the catalytic site

Question 15

name the 2 parameters of michaelis menten enzyme kinetics

Answer 15

Vmax and Km

Question 16

what is Vmax

Answer 16

maximal rate of catalysis given saturating amounts of enzyme
depends on amount of enzyme and how fast it can work
it is the turnover number - enzymatic cycles per second at top speed
plateaus - when saturated

Question 17

what is Km

Answer 17

substrate concentration that supports a rate of catalysis equal to 1/2 Vmax
depends on/is measure of affinity of enzyme substrate binding

Question 18

how does 4 times as much enzyme affect the Vmax and Km

Answer 18

increases Vmax proportionally
Km is same tho (due to binding affinity)

Question 19

what does binding affinity depend on

Answer 19

only the chemical properties of the enzyme and substrate
Independent of concentrations

Question 20

name example of enzyme substrate binding and catalytic site details

Answer 20

serine protease trypsin

Question 21

explain what happens in serine protease trypsin - what it is

Answer 21

proteases hydrolyze peptide bonds in polypeptides
serine proteases are a family of proteases whose catalytic mechanism involves a serine residue in the catalytic site

Question 22

explain what happens in serine protease trypsin - what trypsin does

Answer 22

hydrolyzes peptide bonds adjacent to arginine and lysine (large basic side chains)
proper binding occurs when substrate amino acid side chain fits into negatively charged pocket within substrate binding site

Question 23

what defines differing specificities (of substrate recognition)

Answer 23

differences in the substrate recognition pocket in related enzyme defines their differing specificities

Question 24

describe pocket in elastase

Answer 24

obstructed by bulky valine side chains - so elastase cleaves adjacent to aas with small side chains
eg - alanine and glycine

Question 25

trypsin is an example of what

Answer 25

participation of key catalytic side aa side chains in catalytic mechanism protein folding brings distant aas into proximity ex = trypsin - 2 step catalytic mechanism involving 3 aa side chains (not closely positioned in linear protein sequence)

Question 26

name the 3 aa side chains involved with trypsin catalytic mechanism

Answer 26

Asp-102 His-57 Ser-195

Question 27

describe first step of trypsin catalytic mechanism

Answer 27

cleavage of peptide bond with formation of covalent substrate-enzyme complex Ser195 acyl enzyme

Question 28

describe second step of trypsin catalytic mechanism

Answer 28

hydrolysis of acyl enzyme complex (ester bond between substrate and enzyme)

Question 29

what do both sub- reactions depend on of trypsin catalytic mechanism

Answer 29

His-57's ability to bind and to release proton reactions best happen at a pH near the pK of His ~ pH 7

Question 30

what do enzymes exhibit

Answer 30

pH optima

Question 31

what can pH optima of enzymes reflect

Answer 31

active site acid base chemistry sensitivity of overall protein conformation to charge distribution

Question 32

describe chymotrypsin - what it depends on...

Answer 32

depends on acid base reaction between 2 key active side residues

Question 33

what happens to chymotrypsin reaction below pH 7

Answer 33

reaction cannot occur chymotrypsin has little activity below pH 7

Question 34

describe other proteolytic enzymes (lysosomal hdyrolases) - pH changes

Answer 34

evolved different catalytic mechanism - so they can be active in milieu of lysosomes which has low pH

Question 35

describe chymotrypsin above pH 9

Answer 35

conformation of chymotrypsin disrupted because structurally important amino groups become unprotonated and uncharged so it has little activity

Question 36

what happens to enzymes in a common pathway

Answer 36

often physically associated with one another - either by direct binding interactions or by binding to a common scaffold protein

Question 37

what can evolution do to different enzymes

Answer 37

combine them into a single polypeptide - a multifunctional enzyme

Question 38

name an example of a multifunctional protein

Answer 38

mammalian fatty acid synthase polypeptide contains 7 different active sites catalyzing sequential steps in fatty acid synthesis

Question 39

what are allosteric effects

Answer 39

binding of ligand at one site on a protein can lead to conformational changes that affect the binding of another ligand molecule at a different site

Question 40

where are allosteric effects used majorly

Answer 40

conformational switches in regulatory proteins in response to ligand binding or post translational modification

Question 41

explain an example of allosteric effects

Answer 41

nature of the ligand bound to the nucleotide binding domain of the molecular chaperon - Hsp70 ATP vs ADP (atp = open conformation, but if hydrolyzed to adp shape changes and its a diff interaction), affects the conformation of the substrate binding domain - which changes its interactions with the misfolded client protein

Question 42

what is used as allosteric switches to control protein activity

Answer 42

noncovalent binding of Ca++ and GTP (important in cellular signal transduction pathways)

Question 43

describe Ca++ binding to calmodulin (allosteric effects)

Answer 43

changes its conformation allows it to bind to target peptides on other proteins - thus regulating their structure and activity turns on - makes surface where calmodulin can bind to target peptide

Question 44

describe GTP activity (allosteric effects)

Answer 44

when G proteins are bound to GTP = on conformation when G proteins are GDP bound = off conformation interact slightly differently with other proteins

Question 45

describe how G proteins switch from on to off (GTP activity- allosteric effects)

Answer 45

switching from on to off - facilitated by GAPs switching from off to on - facilitated by GEFs

Question 46

what is GEF (allosteric effects) + function

Answer 46

guanine nucleotide exchange factor must drop GDP - reaction stimulated by GEF and helps system turn on again

Question 47

what is GAP (allosteric effects) + function

Answer 47

GTPase activating protein binds G protein and stimulates hydrolysis of GDP to help turn system off

Question 48

describe how modification of protein conformation and activity can be done

Answer 48

through phosphorylation and dephosphorylation

Question 49

what is a post translation modification

Answer 49

phosphorylation of amino acid side chains a rapidly reversible covalent modification of protein structure

Question 50

when is phosphorylation and dephosphorylation used

Answer 50

very important for regulatory mechanisms used in cellular transduction pathways ex = human genome codes for over 500 different protein kinases - many are specific for just one or a few specific target proteins

Question 51

describe process of phosphorylation and dephosphorylation

Answer 51

target protein with OH can be phosphorylated (comes from ATP) inactive state = not phosphorylated active state = now can do something that it couldn’t before, able and ready to work, has impact on shape of protein *change in conformations between inactive and active states

Question 52

describe cascade effect (reversible protein phosphorylation)

Answer 52

often the target of a kinase or a phosphatase is another kinase or phosphatase (just keeps attacking and amplifying each time creates cascade effect kinase cascade permits amplification of signs, and many levels of fine tuning