Lecture 11

Question 1

What are the 3 interpretations of the Michaelis - Menten curve

Answer 1

– at very low S, S «< Km, then Vo = Vmax [S]/ Km

– at S = Km, Vo = Vmax/2

– At very high [S], [S]&raquo_space;> Km, then Vo = Vmax and rate is independent of [S]

Question 2

How does one determine Km and Vmax accurately ?

Answer 2

– X-intercept: -1/Km
Y- intercept: 1/V max

Slope: Km/Vmax

Question 3

T or F, Km is a concentration value and is always positive.

Answer 3

True

Question 4

What are the 2 types of inhibition?

Answer 4

– Reversible (on-off binding, noncovalent)

– Irreversible (very tight binding to enzyme, covalent)

Question 5

Amongst reversible inhibitors what are the 3 modes of action? And there effect on Km and Vmax

Answer 5

• Competitive: This increases Km but no effects on V max
• Uncompetitive: Decrease both Km and Vmax
• Mixed: Decrease Vmax but increases Km (no competition = no change in Km)

Question 6

Describe competitive inhibition

Answer 6

– Enzyme binds Substrate or Inhibitor (not both)

– Enzyme can be freed from I by increasing S

• • Km is increased (need more S to reach Vmax/2)
• • V max is unchanged

Question 7

What is Ki?

Answer 7

It is the dissociation constant of the inhibitor

Question 8

What is an example of a competitive inhibitor drug?

Answer 8

– the inhibitor of angiotensisn converting enzyme (ACE)

– used in treatment of hypertension

– competitive inhibitor, captopril, bound in active site and to Zn2+

– binds more tightly than angiotension I

– structural analog to angiotension I, normal substrate, which is cleaved by enzyme and released angiotension ii which then elevates BP

Question 9

Describe noncompetitive inhibition

Answer 9

– I binds to ES complex only

– I binding possible only after S binding

– ESI (enzyme substrate inhibitor) complex cannot make P

– Vmax cannot be attained and is lower

– Km also lower (proportionally to Vmax), need less S to reach Vmax/2

– High [S] does not overcome inhibition

Question 10

Describe Mixed inhibition.

Answer 10

– Enzyme can bind inhibitor and Substrate at the same time

– ESI complex cannot make P

– I must dissociate for catalysis to occur

– Vmax is lower

– Km is increased or same (non-comp)

– High S does not overcome inhibition

Question 11

Describe Irreversibile inhibition; and what is an example?

Answer 11

– combine covalently with the enzymes to inactivate them

– many are transition state analogs –> meaning similar to transition state in structure

– binds very strongly to enzyme (tighter binding than substrate)

Ex: DFP binds to active site serine of enzyme, acetylcholinesterase, which is necessary for nerve conduction and causes paralysis of vital functions, very toxic substance

Question 12

T or F, regulation is important for enzyme activity because it doesn’t work to have all enzymes operating at peak activity all the time

Answer 12

True

Question 13

What are the 3 ways Enzyme activity is regulated?

Answer 13

– Substrate level control

– Feedback control

– Allosteric Enzymes

Question 14

Describe Substrate Level Control.

Answer 14

– direction interaction of substrate and products with enzyme

– high levels of substrate can increase reaction rate

– however, high levels of product also compete for binding to enzyme and inhibit formation of more product

Question 15

T or F, Enzymes that are subjected to tight regulation often catalyze “committed steps” along specific metabolic pathway

Answer 15

True; committed step is basically irreversible

Question 16

Describe feedback regulation.

Answer 16

– Enzymes that are subjected to tight regulation often catalyze “committed steps” along specific metabolic pathway

–> committed catalytic step is basically irreversible

–> product of a committed step will essentially continue down pathway to final product

–> targeting first committed catalytic step along a enzymatic pathway helps regulate end product formation without unnecessary loss of energy

Question 17

Describe Allosteric Enzymes.

Answer 17

– Enzymes regulated by allosteric control (allosteric enzymes) are multi-subunit proteins

– Regulation of catalytic (Kcat) and binding activities generally done by inducing significant conformational changes effected by:

–> substate itself at catalytic sites (cooperativity between multiple catalytic subunits); Homoallosteric effect

–> By other ligands at sites distinct from catalytic site to increase or decrease enzymatic activity. Heteroallosteric effect.

Question 18

T or F, Allosteric controls often involved in rapid switching between a highly active catalytic state (R) and a less active state (T) of enzyme

Answer 18

True; useful for quick responses to rapid metabolic changes and needs

Question 19

Describe Homoallosteric effects.

Answer 19

– a single-site M-M enzyme will have a hyperbolic curve, where as multisite binding enzyme as a sigmoidal activity curve

– any enzyme that binds cooperatively will behave at low concentrations as if it binds poorly; then when S increases and more substrate binds, the enzyme is more effective because binding is better

– then enzyme undergoes a transition from lower affinity state (T state) to higher affinity state (R)

Question 20

T or F; with an enzyme that binds cooperatively, it is slow to being but over time speed increases

Answer 20

True

Question 21

T or F; Allosteric enzymes do not follow Michaelis- Menten kinetics

Answer 21

True; curve is different

Question 22

Describe cooperative binding and the differences in substrate concentration.

Answer 22

– cooperative binding: binding of 1 substrate lead to increased binding affinity for substrate and higher catalytic activity at other active sites on an allosteric enzyme

– High [S] : High binding affinity for substrate and high catalytic rates

– INcrease in [S] leader to a sharp increase in catalytic rate (high slope) until maximum rate is reached

– Low [S]: low binding affinity for substrate and low catalytic rate –> has to wait for substrate to bind

Question 23

T or F, heteroallosteric effectors regulate transtition from T to R state at the same site as active site

Answer 23

False; regulate transition at a different site than active site (hetero)

Question 24

T or F, allosteric effectors are only inhibitors of enzymes.

Answer 24

False, they are also activators

Question 25

Describe allosteric enzyme reaction rates with modulators.

Answer 25

– increase or decrease the binding affinity of enzyme for its substrate

– an enzyme that exists in two conformation state (T or R), can have it’s kinetics controlled by another molecule that shifts the equilibrium from one to the other state

– Inhibitors favor T state, and Activators favor R state

Question 26

T or F, Inhibitors favor R state, and Activators favor T state

Answer 26

– False, it is the other way around

Question 27

How is ATCase an example of an allosteric enzyme?

Answer 27

– allosteric enzyme involved in a committed step (step#2) of a multistep enzymatic pathway leading to biosynthesis of pyrimidines nucleotides (building block of nucleic acid + rile in enzyme activity)

Question 28

How is ATCase regulated by ATP and CTP?

Answer 28

– in terms of kinetics, CTP makes it more difficult for bound substrates to convert ATCase to R state and thus makes it more difficult to produce more product

– if CTP is high, we don’t need more produced through pyridimine biosynthesis

– on other hand, hight ATP signals a purine-rich state, which signals a need for pyrimidine biosynthesis (CTP)

– an energy-rich state, where a cell can devote energy to nucleotide biosynthesis

Question 29

What is the structure of ACTase?

Answer 29

• • has 12 subunits
• • 6 catalytic chains organized in 2 trimers (3 active sites/trimer)
• • 6 regulatory chains organized in 3 dimers (where heteroallosteric molecules bind)

– many contacts between regulatory dimer and catalytic trimers

Question 30

Describe Feedback inhibition by CTP

Answer 30

– CTP acts by modulating the equilibrium between ATCase T and R states

–> CTp binds to regulatory subunits preferentially in T state

–> CTP stabilizes low catalytic efficiency T state

–> CTP shifts the equilibrium toward T state

–> CTP decreases the affinity of ATCase for substrates carbamoyl phosphate and aspartate

Question 31

Understanding ATCase catalysis from its structure

Answer 31

– binding of substrate to catalytic sites of ATCase = large conformation changes of ATCase –> not permanent attachments

– T state of ATCase: “tense” compact form with low catalytic activity

– R state of ATCase: “relaxed” expanded form with high catalytic activity

– Substrate changes equilibrium between T and R states toward R state

Question 32

Describe the different cooperative effects (Low, High)

Answer 32

Low [S] = Tstate&raquo_space;> Rstate, lower binding affinity for S, low activity

High [S] = Rstate&raquo_space;> T state, higher binding affinity for S, high activity

– Steep rate increase within narrow changes in [S] due to conformational change