KH5 Flashcards

1
Q

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

A

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

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2
Q

what is a ligand

A

molecule that a protein binds to

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3
Q

what happens when ligand binds sometimes

A

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

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4
Q

name the 2 important properties in ligand binding

A

specificity
affinity

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5
Q

what is specificity

A

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

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6
Q

what is affinity

A

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

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7
Q

what is protein binding

A

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

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8
Q

describe a stable complex vs less stable complex in protein binding

A

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

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9
Q

describe antibody binding properties

A

bind antigens with high specificity and high affinity

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10
Q

describe how antigens and antibodies bind

A

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

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11
Q

what are enzymes

A

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

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12
Q

where do substrate binding and reaction catalysis happen

A

at enzymes active site

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13
Q

what does substrate specificity arise from

A

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

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14
Q

what do aas contribute to (substrates/enzymes)

A

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

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15
Q

name the 2 parameters of michaelis menten enzyme kinetics

A

Vmax and Km

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16
Q

what is Vmax

A

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

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17
Q

what is Km

A

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

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18
Q

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

A

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

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19
Q

what does binding affinity depend on

A

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

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20
Q

name example of enzyme substrate binding and catalytic site details

A

serine protease trypsin

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21
Q

explain what happens in serine protease trypsin - what it is

A

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

22
Q

explain what happens in serine protease trypsin - what trypsin does

A

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

23
Q

what defines differing specificities (of substrate recognition)

A

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

24
Q

describe pocket in elastase

A

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

25
trypsin is an example of what
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)
26
name the 3 aa side chains involved with trypsin catalytic mechanism
Asp-102 His-57 Ser-195
27
describe first step of trypsin catalytic mechanism
cleavage of peptide bond with formation of covalent substrate-enzyme complex Ser195 acyl enzyme
28
describe second step of trypsin catalytic mechanism
hydrolysis of acyl enzyme complex (ester bond between substrate and enzyme)
29
what do both sub- reactions depend on of trypsin catalytic mechanism
His-57's ability to bind and to release proton reactions best happen at a pH near the pK of His ~ pH 7
30
what do enzymes exhibit
pH optima
31
what can pH optima of enzymes reflect
active site acid base chemistry sensitivity of overall protein conformation to charge distribution
32
describe chymotrypsin - what it depends on...
depends on acid base reaction between 2 key active side residues
33
what happens to chymotrypsin reaction below pH 7
reaction cannot occur chymotrypsin has little activity below pH 7
34
describe other proteolytic enzymes (lysosomal hdyrolases) - pH changes
evolved different catalytic mechanism - so they can be active in milieu of lysosomes which has low pH
35
describe chymotrypsin above pH 9
conformation of chymotrypsin disrupted because structurally important amino groups become unprotonated and uncharged so it has little activity
36
what happens to enzymes in a common pathway
often physically associated with one another - either by direct binding interactions or by binding to a common scaffold protein
37
what can evolution do to different enzymes
combine them into a single polypeptide - a multifunctional enzyme
38
name an example of a multifunctional protein
mammalian fatty acid synthase polypeptide contains 7 different active sites catalyzing sequential steps in fatty acid synthesis
39
what are allosteric effects
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
40
where are allosteric effects used majorly
conformational switches in regulatory proteins in response to ligand binding or post translational modification
41
explain an example of allosteric effects
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
42
what is used as allosteric switches to control protein activity
noncovalent binding of Ca++ and GTP (important in cellular signal transduction pathways)
43
describe Ca++ binding to calmodulin (allosteric effects)
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
44
describe GTP activity (allosteric effects)
when G proteins are bound to GTP = on conformation when G proteins are GDP bound = off conformation interact slightly differently with other proteins
45
describe how G proteins switch from on to off (GTP activity- allosteric effects)
switching from on to off - facilitated by GAPs switching from off to on - facilitated by GEFs
46
what is GEF (allosteric effects) + function
guanine nucleotide exchange factor must drop GDP - reaction stimulated by GEF and helps system turn on again
47
what is GAP (allosteric effects) + function
GTPase activating protein binds G protein and stimulates hydrolysis of GDP to help turn system off
48
describe how modification of protein conformation and activity can be done
through phosphorylation and dephosphorylation
49
what is a post translation modification
phosphorylation of amino acid side chains a rapidly reversible covalent modification of protein structure
50
when is phosphorylation and dephosphorylation used
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
51
describe process of phosphorylation and dephosphorylation
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
52
describe cascade effect (reversible protein phosphorylation)
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