Non Michaelis-Menten Kinetics

Question 1

What particularly about the xenobiotic metabolizing enzyme do we focus on in Biochemical Pharmacology?

Answer 1

‘’Unusual’’ in vitro kinetic behaviour

Question 2

What are the two main members under xenobiotic metabolizing enzymes?

Answer 2

Cytochrome P450 (CYP) enzyme system and UDP-glucuronosyl transferase (UGT) enzymes

Question 3

What is autoactivation?

Answer 3

It results in an initial lag in the rate-substrate concentration profile generating a sigmoidal curve

Question 4

What is autoinhibition?

Answer 4

It is characterized by a convex curve due to Vmax not being maintained at high substrate concentrations

Question 5

What is the problem with the Michaelis-Menten model?

Answer 5

There are particular kinetic features for several CYP and UGT substrates that the M-M model cannot explain

Question 6

What is cooperative binding?

Answer 6

Cooperative binding is when one substrate binds to make the next substrates easier to bind

Question 7

What kind of enzyme model is expected from a Non-M-M kinetics?

Answer 7

Enzyme models with multiple sites showing cooperative binding for the drug substrate

Question 8

Define allosterism

Answer 8

Allosterism is “binding at an alternative site

Question 9

True or False: Allosterism in vivo exists

Answer 9

False: allosterism in vivo does not exist, but it does in an in vitro setup where it can either be co-operative or inhibitory

Question 10

What is S50 and what is it analogous to?

Answer 10

S50 the 50% of the Vmax and it is analogous to the Km from the Michaelis-Menten equation

Question 11

DOUBKE CHECK: What is n referring to in the Hill Model equation?

Answer 11

n is the unique Hill coefficient

Question 12

Describe what 1B is by uncompetitive inhibition

Answer 12

v = Vmax/(1+(Km/S)+(S/Ksi))

Question 13

Describe a substrate inhibition curve

Answer 13

Substrate inhibition curves are concentration-dependent: low concentration looks like M-M and in higher substrate concentration, the curves go “off piste”

Question 14

What are Ks and Kp?

Answer 14

Ks is the substrate dissociation constant and Kp is the catalytic rate constant

Question 15

What happens when α < 1?

Answer 15

The binding affinity for the second substrate molecule is increased (co-operative binding), which enhances the overall product formation rate and results in autoactivation

Question 16

What is the change in Kp by the factor β?

Answer 16

It is when both substrate sites are occupied. This change is an alternative mechanism for autoactivation

Question 17

What happens when β > 1?

Answer 17

The overall rate of the reaction is increased (autoactivation)

Question 18

What happens when β < 1?

Answer 18

The overall rate is decreased (autoinhibition)

Question 19

What can the model/kinetic scheme for substrate interactions at two separate binding sites be used for?

Answer 19

The model can be used to describe data from substrates showing both sigmoidicity/co-operation and substrate inhibition

Question 20

True or False: there are no direct relationships between parameters from the model (model/kinetic scheme for substrate interactions at two separate binding sites) and the Hill coefficient, n, or the substrate inhibition, Ksi

Answer 20

True

Question 21

How can a positive cooperative effect or sigmoidicity be observed?

Answer 21

When a value of α < 1 and β > 1

Question 22

Which two conditions can observe a negative cooperative effect?

a. value of α > 1, resulting in a biphasic kinetic profile
b. value of α < 1, resulting in a biphasic kinetic profile
c. value of β > 1, causing substrate inhibition
d. value of β < 1, causing substrating inhibition

Answer 22

a. value of α > 1, resulting in a biphasic kinetic profile
and
d. value of β < 1, causing substrating inhibition

Question 23

What problem do these models have that you need to be aware of?

Answer 23

The models do not distinguish between the simultaneous binding of multiple molecules within a single active site and the binding of two molecules at two distinct sites

Question 24

How does in vitro-in vivo scaling strategy work?

Answer 24

This strategy is used to describe fully the in vitro data, and then abstract something useful for extrapolation to real human or animal drug research

Question 25

Why is it important to comprehensively characterize in vitro systems?

Answer 25

To know and understand the limits that may extend beyond those observed in vivo

Question 26

Why are substrate and product inhibition unlikely to be of consequence in vivo?

Answer 26

Due to the high concentrations required and because clearance is constantly occuring

Question 27

What issue arises from forcing Michaelis-Menten kinetics in in vivo and/or in vitro setups?

Answer 27

Sigmoidal or inhibitory kinetics that may or may not be functions of the test tube system are not recognized and accounted for, which can affect what will happen in the human body due to inappropriately analyzed data

Question 28

What is the minimum value for H in a CLmax equation and what does it correspond to?

Answer 28

Minimum of 0.5 H value that corresponds to n = 2 substrates binding

Question 29

What are some of the processes that would lower the [Substrate] available to the enzyme relative to the concentration calculated, after the addition of a particular [Substrate] to the test tube?

Answer 29

1. Substantial substrate depletion
2. Saturable futile binding of substrate or metabolites (products) within the incubation matrix
3. Saturable cellular efflux processes - in a whole cell culture system, transporters, especially ABC transporters may be pumping drug or product/metabolite out
4. Poorly defined limit of analytical quantitation (concerns with the instrument used)
5. Aqueous solubility limitations (precipitation of drug or metabolites)

Question 30

What is the impact on processes that involve saturable events?

Answer 30

It would result to concentration-dependence (maximal at the low concentrations and tapering off to no effect at high concentrations)

Question 31

What is the ideal turnover of substrate?

Answer 31

10% or less (<10%) to get sigmoidicity and to comply with initial rate conditions and prevent product inhibition

Question 32

What is the role of active transporter systems?

Answer 32

To detect any inconsistency between cellular and media concentrations of drug substrates

Question 33

If cellular efflux is significantly different than the Km for metabolism, sigmoidicity could ______

Answer 33

arise

Question 34

What are some of the consequences of ignoring sigmoids?

Answer 34

1. Problems with first in human or first in animal drug trials (oversight)
2. Underprediction of CLint (calculated drug dose is sub-therapeutic)
3. Underestimation of Vmac for substrate inhibition will occur by ignoring the high concentration data points and forcing a M-M model through the remaining lower [substrate] data (Km is poorly estimated)
4. Bad choice of model to estimate clearance gives poor quality data points (not rich enough)

Question 35

What is the problem with many Pharma/Biotech investigators?

Answer 35

They only work with one substrate concentration and asses inhibitory potential by the concentration responsible for a 50% inhibition value (I50)

Question 36

How is the interpretation of the nonhyperbolic type behavior complicated?

Answer 36

Effects are not consistently seen with all substrates for the same enzyme as they vary by substrate or drug