Understanding Allosteric & Competitive Enzyme Inhibitors

[physicisttobe]

Homework Statement

Enzyme kinetics

Relevant Equations

no

Hi everyone!

My question has something to do with biochemistry. Therefore, I ask it here because biochemistry is a part of chemistry.

My question is: how can you find out whether an enzyme inhibitor acts allosterically or competitively?

My answer to this is: if the active site of the enzyme is blocked by an inhibitor, then it is competitive. If any other side of the enzyme (non-active side) is blocked, then it is allosteric. We also discussed that the relative substrate concentration decreases when we have competitive inhibitor, but why? Why does the substrate concentration decrease? The concentration remains the same, doesn't it? Because we have an inhibitor and this binds to the active side of the enzyme, so the concentartion does not change, nothing happens with it. Why does the relative substrate concentration change??

And also the km-value describes us, if it is competitive or non competitive.

 

[epenguin]

Homework Statement:: Enzyme kinetics
Relevant Equations:: no

Hi everyone!

My question has something to do with biochemistry. Therefore, I ask it here because biochemistry is a part of chemistry.

My question is: how can you find out whether an enzyme inhibitor acts allosterically or competitively?

My answer to this is: if the active site of the enzyme is blocked by an inhibitor, then it is competitive. If any other side of the enzyme (non-active side) is blocked, then it is allosteric. We also discussed that the relative substrate concentration decreases when we have competitive inhibitor, but why? Why does the substrate concentration decrease? The concentration remains the same, doesn't it? Because we have an inhibitor and this binds to the active side of the enzyme, so the concentartion does not change, nothing happens with it. Why does the relative substrate concentration change??

And also the km-value describes us, if it is competitive or non competitive.

I'll try bit by bit because there are some confusions possibly getting in your way here.

'Competitive' and 'allosteric' are not opposing terms. They refer to different aspects of the interaction. Fully competitive is when either substrate or inhibitor but not both can be bound to an enzyme. 'Allosteric' is about where substrate and inhibitor are bound - if at the same site the inhibition is called 'isosteric', if at a different site this is called allosteric, the site is called the allosteric site. There is no reason an allosteric site cannot be still competitive in the sense defined above, and indeed it often is.

I just don't know what you mean by 'relative substrate concentration decreases'..., maybe you need to re-read your text or notes. For your last sentence there is indeed a thing called the "apparent K_m" of substrate which does increase as inhibitor concentration increases in the case of competitive kinetics. But then K_m refers to the Michaelis equation, and allosteric enzymes most often do not obey that equation. One often then uses descriptively the
[S ]_½ (substrate concentration at half maximal velocity).

You ask how can allosteric binding be distinguished from isosteric, and I don't know that there is any formal general method other than obtaining by x-ray crystallography etc the structures of the enzyme and its complex with substrate close substrate analogues and with the allosteric effector. The term 'Allosteric' was first introduced before any such structures were known, because although there was a predisposition to imagine everything was competition for the same site, an accumulation of mainly indirect evidence was showing this was hard to believe.

 

[physicisttobe]

First of all thank you so much for your reply and for your explanation!

In the slides it is described that the relative substrate concentration decreases and I don't know what is meant by that. However, the substrate concentration remains the same, right? This does not decrease, right? My professor said that the substrate concentration decreases and I don't understand this. Do you have any idea what that means, or what do you think they mean by that?

I have a question about isosteric binding: how can it be that the substrate and the inhibitor both attack the same site at the same time?

 

[epenguin]

Sorry those words "relative substrate concentration decreases" by themselves don't mean anything, can you explain further, like when, relative to what?

in isosteric interaction they can't both occupy the site at the same time, that's how the isosteric inhibitor inhibits - occupying a catalytic site that would otherwise be available to the substrate.

 

[physicisttobe]

In the slides it is explained like this:

competitive inhibitor:
- competes with the substrate for the active site of E (enzyme)
- relative substrate concentration decreases 
- KM ↑ , vmax = constant

non-competitive inhibitor:
- binds outside the active site of E and ES
- concentration of active sites decreases
- vmax ↓ , KM = constant

So I do not understand this explanation. Why does the substrate concentration decrease, maybe there is a mistake, but my prof. also told that the concentration decreases.

 

[epenguin]

I think the phrase "relative substrate concentration decreases" does not mean anything and you can safely ignore it. Rest of the slides are OK.

When I mentioned indirect evidence previously the kind of thing is desensitisation – sometimes by heating or other denaturation treatment an enzyme could be made to lose the inhibition susceptibility whilst still retaining the catalytic activity. Then as well as allosteric inhibition inhibition there is allosteric activation. It is a bit hard to imagine how you could have steric activation.

 

[physicisttobe]

All right! Thank you so much for all your explanations!

Can I ask further questions here in this forum about biochemistry? There is no subforum for biochemistry, so I was not sure if it`s appropriate to ask questions about it. I´m studying for my biochemistry exams and it is quite helpful when I can ask my questions here and discuss with you about specific topics.

 

[berkeman]

Can I ask further questions here in this forum about biochemistry? There is no subforum for biochemistry, so I was not sure if it`s appropriate to ask questions about it. I´m studying for my biochemistry exams and it is quite helpful when I can ask my questions here and discuss with you about specific topics.

Yes, for schoolwork questions, the Homework Help forums are the correct area of PF to post. And this Biology and Chemistry subforum in HH is the best match for your biochemistry questions.

Please start a new thread for each different question to avoid confusion, and show as much of your work as you can -- you should get great help as long as you show your efforts. Thanks.

 

[physicisttobe]

I am very pleased to hear that, thanks a lot :)

 

[physicisttobe]

@epenguin, I have given some further thought to this qustion "how can you find out whether an enzyme inhibitor acts allosterically or competitively?"
I have an idea how to figure out whether an enzyme inhibitor is allosteric or competitive. I hope that my reasoning is correct:
If the Km value changes and vmax stays the same, then it is a competitive. If the Km value
changes, but vmax remains unchanged then it acts allosterically. You also have a graph/curve where you can see whether it is competitive or allosteric. Maybe we can justify it in this way. Do you think that this justification is correct?
This is a really important question, therefore I would like to find an appropriate answer.

 

[epenguin]

You are ignoring what I already tried to explain: 'Allosteric' and 'competitive' are not opposites, and K_m is not quite the appropriate parameter for the affinity of a substrate that does not obey the Michaelis equation.

If the [S ]_½ is increased by the inhibitor but V_max unaffected (and increase in substrate concentration increases [I ]_½) then their interaction is of competitive nature, that is the how. The pair might be found at the same site or different, isosterically or allosterically, that is the where.

Cases where only substrate affinity is changed by the inhibitor are called 'K systems', those where only V_max 'V systems'.

I don't know offhand what reading to recommend, but probably any of the big biochemistry textbooks. I think the terminology was originally given by Monod Changeux & Jacob in a paper in the Journal of Molecular Biology 6 309-329 (1963).

It is the right time where I am to wish you the best for the coming year!

 

[DonaldGreens]

Also an interesting and exciting topic! Which has interested me for a long time! Almost got all the answers to my questions!

 

[physicisttobe]

@epenguin, thanks four your explanations! I will think about it and make some further notes. If some questions arise, I´ll come back to you.

@DonaldGreens, that sounds great! I'm glad for you :)

 

[epenguin]

@epenguin, thanks four your explanations! I will think about it and make some further notes. If some questions arise, I´ll come back to you.

That is a good attitude. It is possible to question some statements or assumptions in this field.

I thought it might be helpful to point out to you the publication where the term "allosteric" was first introduced (#11). If you read this, bear in mind when it was written the 3-D structure of not a single enzyme was known, let alone that of an enzyme-inhibitor complex. At that time to explain their rather indirect experimental findings, enzymologists might timidly advance hypothetical or schematic mechanisms and schematic partial structures. If you could invent an extra site for everything that affected enzyme catalysis it seemed you could explain everything and nothing, so they restrained themselves from doing this. Just Monod et al. were pointing out in this publication that even so, the evidence of thelse "allosteric effects" was hard to resist.

Although you might question some of what is said about it you also have to know something about what this is. I don't know if it is clear to you that in classical competitive inhibition you are inferring from experimental kinetic findings that an enzyme can form a complex with a given substrate "ES", or with an inhibitor "EI", but not with both "ESI" at the same time. That in itself says nothing about S and I being bound at the same place or not.

 

[physicisttobe]

Thanks a lot for your explanation. I understood it better now.