Module 5

Question 1

ES & EP are ___

Answer 1

stable species that would have appreciable concentrations & could be isolated (are intermediates)

Question 2

What are transition states?

Answer 2

transient species that form along the reaction pathway - are found at the top of peaks in free energy plots

Question 3

When do the maximum interactions between S & E occur?

Answer 3

when S reaches the transition state, when the free energy (binding free energy) released by the S-E interactions partly overcomes the energy needed to get to the ‘top of the hill’

Question 4

Enzymes reduce the activation energy & accelerate rates of reactions by. . .

Answer 4

1) binding substrates in the correct orientation relative to the active groups

2) providing catalytically active groups (side chains, acids, bases, metal ions)

3) polarizing bonds, stabilizing charged species (usually unstable)

4) stabilizing the transition state

Question 5

Why does RR decrease with time?

Answer 5

1) S is depleted by conversion to product

2) reaction is REVERSIBLE, so as [P] increases, the rate of REVERSE reaction increases

3) enzyme may be unstable under the reaction conditions

Question 6

The rate of an enzyme catalyzed reaction is proportional to. . .

Answer 6

enzyme concentration

Question 7

Describe the features of a Michaelis-Menten plot

Answer 7

RECTANGULAR HYPERBOLA

• asymptote = Vmax

Question 8

What is Km? (Michaelis constant)

Answer 8

substrate concentration at which we reach halfway to Vmax

Question 9

What are the 4 assumptions of the Michaelis Menten theory?

Answer 9

1) ES conversion to E+P is irreversible

2) Steady-state conditions [ES] constant (the rate of formation of ES is that same as the rate of breakdown of ES)

3) [S]&raquo_space; [Et] should have way more substrate than enzyme

4) [S]&raquo_space; [P] initial conditions at least 10 fold higher

Question 10

What are irreversible inhibitors?

Answer 10

bind COVALENTLY to active site, destroy a functional group, essential for enzyme activation or form a stable non-covalent complex with the enzyme (suicide inhibitors)

Question 11

What are reversible inhibitors?

Answer 11

bind reversibly to enzymes and inhibit the enzyme either by COMPETITIVE, UNCOMPETITIVE, OR MIXED modes of inhibition

Question 12

Where does a competitive inhibitor bind?

Answer 12

• in binds to free enzyme; the effective concentration of free enzyme drops by a (alpha)
• a > 1
• the BIGGER alpha, the MORE we’ve dropped the free enzyme concentration

Question 13

Lineaweaver-Burk analysis for COMPETITIVE INHIBITION

Answer 13

• Vmax does not change
• the magnitude of the intercept gets smaller, so Km looks like it gets bigger
• the apparent Km is bigger in PRESENCE of inhibitor

looks like enzyme is binding substrate more weakly (it isn’t really)

Question 14

How does an uncompetitive inhibitor bind?

Answer 14

• binds to the ES complex to form ESI
• has no activity and cannot turn over product
• KI’
• causes a decrease in effective concentration of ES complex

Question 15

Lineaweaver-Burk analysis of uncompetitive inhibition

Answer 15

• increases x and y intercepts
• apparent Vmax DECREASES
• apparent Km gets smaller (looks like it binds the enzyme more tightly)

Question 16

How does a mixed inhibitor bind?

Answer 16

can bind to BOTH free enzyme & ES complex

• binding to enzyme is described by equilibrium constant (KI), whereas binding of inhibitor to ES complex is described by KI’

Question 17

Lineweaver-Burk analysis for mixed inhibition

Answer 17

• no intersection at y-axis
• as we increase inhibitor concentration, we increase value of alpha and alpha’
• apparent Vmax gets smaller
• Magnitude of x-intercept gets smaller

Question 18

• In mixed inhibition, the apparent Vmax . . .

Answer 18

always DECREASES, while the apparent Km can either increase or decrease depending on whether the inhibitor binds to the enzyme ALONE or the enzyme-substrate complex

Question 19

What are some features of allosteric enzymes?

Answer 19

• regulate metabolic pathways by CHANGING activity in response to changes in concentration of molecules around them
• allosteric enzymes are regulated by “allosteric modulators” or “allosteric effectors”
• positive modulators ACTIVE, negative modulators INHIBIT allosteric enzymes
• modulators bind reversibly and non-covalently to enzymes

Question 20

Explain how a positive modulator works

Answer 20

• binds to regulatory site and causes CONFORMATIONAL change at active site
• S can now bind with higher affinity
• shifts from T to R state
• dynamic equilibrium b/w these 2 states

Question 21

Negative modulators stabilize. . .

Answer 21

the T state

Question 22

We don’t use Km with allosteric modulators. Instead, we use. . .

Answer 22

K0.5

(because it doesn’t describe Michaelis menten kinetics)

Question 23

What is an example of an allosteric enzyme?

Answer 23

ATCase

catalyzes the first step in the E. coli pathway to produce the nucleotides UTP & finally CTP

when present at high levels, CTP INHIBITS ATCase (negative modulator)

High ATP levels in bacteria is a POSITIVE modulator (indicates cell growth and need for more CTP)

Question 24

Describe the structure of ATCase

Answer 24

• complex quaternary structure of 12 subunits
• 6 catalytic subunits, 2x trimeric complexes
• 6 regulatory subunits, 3x dimeric complexes

ATP = activator

CTP = inhibitor