4. Enzyme Kinetics

Question 1

how do enzymes affect equilibrium?

Answer 1

they dont!! just speeds up the reaction

Question 2

what does a reaction coordinate diagram show?

Answer 2

shows the reaction progress

Question 3

what is the transition state?

Answer 3

barrier of energy that must be overcome to react product

Question 4

what would happen if there was no transition state?

Answer 4

substrate wouldn’t be stable

Question 5

how do enzymes affect transition state?

Answer 5

enzymes allow for a new transition state by lowering the energy barrier

they do this setting up an optimal substate position, weakening the bonds with environment, etc to allow smooth transition from substrate to product

Question 6

what does the speed of enzymatic reaction rely on?

Answer 6

rate of reaction, determined by transition state

Question 7

what does equilibrium depend on?

Answer 7

equilibrium depends on the energy difference between the substrate and product –> not affected by enzyme

Question 8

describe what happens at transition state and how

Answer 8

substrate undergoes structural changes –> bonds breaking and making, unstable intermediate

Question 9

what is activation energy? how do enzymes affect it?

Answer 9

energy required to induce the structural change of the substrate at the transition state

enzymes reduce it –> faster reaction

Question 10

is there a specific direction a molecule is more likely to go thru?

Answer 10

no direction –> equally likely a molecule decays in either direction, i.e. substrate or product

Question 11

what is the active site?

Answer 11

specialized pocket where reaction occurs

Question 12

what is the environment of the active site? why?

Answer 12

hydrophobic to isolate itself from water

Question 13

how does the active site work?

Answer 13

allows reactants to be properly positioned to easily attain their transition state by weakening bonds

(may participate in reaction)

Question 14

does the active site have stronger interaction with transition state, substrate, or product?

Answer 14

transition state

Question 15

what would be the result if an enzyme’s active site was complementary to the substrate?

Answer 15

substrate is too stabilized –> cannot reach transition state and enzyme acts as competitive inhibitor of the transition state –> no drive to catalyze anything

Question 16

what would be the result if an enzyme’s active site was complementary to the transition state?

Answer 16

substrate can fit into enzyme but not perfectly –> can reach transition state naturally but FASTER!

Question 17

What is the induced fit model of binding?

Answer 17

active site approximates the substrate, then properly binds once substrate is there

Question 18

what is the selected fit model of binding?

Answer 18

active site changes conformation constantly and ligand stabilizes 1 conformation

Question 19

describe the equilibrium of enzyme activity

Answer 19

E+S <–binding–> ES <–catalysis–> EP <–release–> E+P

each step is in equilibrium and has diff transition state

Question 20

what do serine proteases do?

Answer 20

cleave peptide bonds

Question 21

describe the 3 components of the serine protease active site

Answer 21

1. Ser –> OH group acts as nucleophile that attacks peptide bond of substrate
2. His –> accepts H from Ser OH to make nucleophile
3. Asp –> H bonds with His, making N more electronegative

Question 22

describe the alkaline phosphatase experiment

Answer 22

to see how fast substrates are consumed by phosphatase

if phosphatase consumes NPP (colourless), will produce NP (yellow) and can measure amount of yellow

Question 23

what happens if you have more substrate per enzyme?

Answer 23

will saturate the enzyme and reach Vmax

Question 24

what are the units of velocity?

Answer 24

mM/min

Question 25

in the curve showing [product] vs time, what happens when the curve flattens?

Answer 25

substrates have been consumed --> enzyme stops working --> plateaus at an amount of products

Question 26

what does the michaelis menten curve show?

Answer 26

velocity vs [substrate] indicates that reaction speed will increase proportionally to [substrate] then saturate at Vmax

Question 27

describe Vmax

Answer 27

independent of [substrate] --> there is more substrate than active sites available so increasing [substrate] has little effect

Question 28

what is Michaelis constant?

Answer 28

Km = [substrate] and 50% Vmax = [substrate] when 50% of active sites are occupied

Question 29

what does a high Km mean?

Answer 29

more substrate is required for a given reaction rate --> enzyme is less efficient, weak binding

Question 30

what does a low Km mean?

Answer 30

less substrate is required for a given reaction rate --> enzyme is more efficient, high affinity

Question 31

what do michaelis-menten equations assume?

Answer 31

assume formation/release of product is irreversible

Question 32

how do you calculate Km?

Answer 32

Km = (k2 + k3)/k1 k1: E+S --> ES k2: ES --> E+S k3: ES --> E+P

Question 33

what is the michaelis menten equation for V0

Answer 33

V0 = (Vmax*[S])/(Km+[S])

Question 34

what is the problem with michaelis-menten plots? what do we use instead?

Answer 34

michaelis-menten plots may not show enough data points to determine whether curve has reached a maximum or not --> Vmax and Km likely inaccurate use Double Reciprocal / Lineweaver-Burk

Question 35

what is the double reciprocal/lineweaver burk plot?

Answer 35

uses reciprocals of velocity and reciprocals of [substrate]

Question 36

how do you find the Vmax in the double reciprocal/lineweaver burk plot?

Answer 36

y-intercept

Question 37

how do you find the Km in the double reciprocal/lineweaver burk plot?

Answer 37

slope or x-intercept

Question 38

do enzymes only have 1 Km value?

Answer 38

no! can diff Km values for diff substrates

Question 39

why may some enzymes have high Km even though it's not efficient?

Answer 39

may be tailored to be slow to regulate certain conditions

Question 40

what is competitive inhibition?

Answer 40

multiple substrates binding same site

Question 41

how does a reaction change with competitive inhibitor?

Answer 41

velocities decrease and Vmax stays the same always, Km increases

Question 42

how does increasing [substrate] affect competitive inhibition?

Answer 42

makes the inhibitor less effective --> outcompetes the inhibitor

Question 43

what is uncompetitive inhibition?

Answer 43

requires substrate for inhibitor to bind and inhibit --> i.e. inhibitor needs ES complex

Question 44

what happens if you increase [substrate] in uncompetitive inhibition?

Answer 44

there is no competition bc they bind diff sites

Question 45

how do Vmax and Km change in uncompetitive inhibition?

Answer 45

both Vmax and Km decrease

Question 46

what is non-competitive inhibition?

Answer 46

inhibitor binds in presence OR absence of substrate bc binds diff sites --> allows control by cell itself

Question 47

how does inhibition change if you increase [substrate] with non-competitive inhibition?

Answer 47

no effect!

Question 48

what is irreversible inhibition?

Answer 48

covalent bonds --> nothing goes on or off

Question 49

what is a suicide inhibitor?

Answer 49

covalently attaches to enzyme and inhibits it --> tricks enzyme into thinking the substrate has bound

Question 50

what is a transition state inhibitor?

Answer 50

mimics catalytic transition state with HIGHER AFFINITY than substrate or product (non-covalent, but quasi irreversible binding)

Question 51

what are the 3 values to compare inhibitor effectiveness?

Answer 51

1. Ki 2. IC50 3. EC50

Question 52

describe Ki

Answer 52

EI <=> E+I Ki = ([E][I]/[EI]) lower Ki = more potent inhibitor

Question 53

describe IC50 and its equation

Answer 53

[inhibitor] that inhibits 50% of enzyme IC50 = Ki + [E]/2

Question 54

describe EC50

Answer 54

[inhibitor] that reduces cellular effect by 50%

Question 55

what is the difference in studying IC50 vs EC50?

Answer 55

IC50 --> can look at pure enzyme + inhibitor EC50 --> must look at whole cell system to see downstream effect