The Effect of Changing the Concentration of an Enzyme...

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The Effect of Changing the Concentration of an Enzyme...

Postby jebus197 on April 5th, 2010, 4:17 pm 

Hi I have to do a biology write up and it seems to be going OK so far. The title of the essay is "The Effect of Changing the Concentration of an Enzyme on the Rate of an Enzyme Catalysed Reaction". Obviously in this case, the increased concentration of enzyme results in an increased rate of reaction, up to a given point when all of the active sites of the enzyme are used up. However the question that occurred to me (and one not asked in the essay) is what would happen if you just kept adding more substrate and simply replenished the substrate as it was used up? In other words, would the enzymes continue to work indefinitely? Would they never stop working? And if they did stop working, at what point would this be and why?
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Re: The Effect of Changing the Concentration of an Enzyme...

Postby Paralith on April 5th, 2010, 4:35 pm 

I assume you meant, that increasing the enzyme concentration does not change the rate of reaction when all the substrate is used up /monopolized, correct? I'm sure there are plenty of enzyme active sites left if you keep adding more enzyme. :)

From my understanding, the enzymes would continue to work until something else causes them to lose their conformation or otherwise degrade, like a change in environment or the arrival of another enzyme designed to break down the previous enzyme. But we have some biochemists here who will know better than I do.
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Re: The Effect of Changing the Concentration of an Enzyme...

Postby MrMistery on April 5th, 2010, 4:47 pm 

An enzyme is a protein that has a specific three-dimensional shape. Without that shape, it cannot do whatever it is that it does. When placed at room temperature, all enzymes will slowly loose this three-dimensional shape. So in your hypothetical experiment, the enzyme will eventually get inactivated, though it would take a very long time for it. Exactly how long this would take would depend on the enzyme. An enzyme from a bacterium that normally functions at high temperature would take longer to misfold, but it would happen eventually.
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Re: The Effect of Changing the Concentration of an Enzyme...

Postby Paralith on April 5th, 2010, 4:54 pm 

MrMistery wrote:An enzyme is a protein that has a specific three-dimensional shape. Without that shape, it cannot do whatever it is that it does. When placed at room temperature, all enzymes will slowly loose this three-dimensional shape. So in your hypothetical experiment, the enzyme will eventually get inactivated, though it would take a very long time for it. Exactly how long this would take would depend on the enzyme. An enzyme from a bacterium that normally functions at high temperature would take longer to misfold, but it would happen eventually.


So even if maintained in ideal conditions for its conformation, an enzyme will inevitably misfold after enough time has gone by - even it's a very very very long time?
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Re: The Effect of Changing the Concentration of an Enzyme...

Postby jebus197 on April 5th, 2010, 5:39 pm 

Paralith wrote:I assume you meant, that increasing the enzyme concentration does not change the rate of reaction when all the substrate is used up /monopolized, correct? I'm sure there are plenty of enzyme active sites left if you keep adding more enzyme. :) .


Yeah you're right, but then the limiting factor becomes the availability of substrate. You can keep adding more enzyme, but unless you add more substrate too, eventually the reaction will be occurring as quickly as possible until all the substrate is used up. (So you would end up with a fairly classic bell curve graph).

So am I right to assume that simply adding more substrate would result in a similar (although perhaps longer, in terms of time and the amount of products produced) type of graph, where eventually (due to denaturing of the proteins at room temperature) the reaction would naturally tail off to zero?
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Re: The Effect of Changing the Concentration of an Enzyme...

Postby MrMistery on April 5th, 2010, 7:38 pm 

@jebus
Well it might depend on the thermodynamics. If you are investigating a reaction such hydrolysis then yes, you are correct, the two graphs would both look optimal (rise to reach a maximum and then decrease to 0). However, that will only go for reactions that are irreversible thermodynamically. If your reaction is actually reversible, then as the product accumulates it will begin to bind the enzyme and the reverse reaction will begin to occur.

@Paralith
What do you mean by "ideal for its conformation"? Thermal energy makes bonds in a protein constantly vibrate, the higher the temperature, the higher the vibration. Theferore, every once in a while, the protein will, by chance, "jump" to a conformation that it can't come back from. Thermostabile proteins are more stably folded, the vibration is more restricted, so the jump happens less often. There is no ideal temperature to retain conformation; basically the colder the better. That's why proteins always need to be kept on ice when working with them and they need to be stored at -80 degrees C.
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Re: The Effect of Changing the Concentration of an Enzyme...

Postby jebus197 on April 5th, 2010, 9:05 pm 

@jebus
Well it might depend on the thermodynamics. If you are investigating a reaction such hydrolysis then yes, you are correct, the two graphs would both look optimal (rise to reach a maximum and then decrease to 0). However, that will only go for reactions that are irreversible thermodynamically. If your reaction is actually reversible, then as the product accumulates it will begin to bind the enzyme and the reverse reaction will begin to occur.


Erm so what's a good (and simple to understand) example of a 'reversible' process?

There is no ideal temperature to retain conformation; basically the colder the better. That's why proteins always need to be kept on ice when working with them and they need to be stored at -80 degrees C.


You mean they need to be stored at this temperature invariably? This is the common practice? (For all proteins/enzymes?)

Why aren't they damaged at such low temperatures?
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Re: The Effect of Changing the Concentration of an Enzyme...

Postby MrMistery on April 5th, 2010, 9:48 pm 

for example an enzyme that isomerizes glucose to fructose. Feed it glucose and fructose will accumulate, but as the fructose accumulates it will be converted back to glucose.

Yes, all proteins need to be stored for a long term at -80. You can store them at -20 if you only plan to keep them for a little while. And they are still damaged by the same thermal agitation, only it takes much longer to misfold at -80 than at room temperature
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Re: The Effect of Changing the Concentration of an Enzyme...

Postby Paralith on April 5th, 2010, 10:36 pm 

MrMistery wrote:@Paralith
What do you mean by "ideal for its conformation"? Thermal energy makes bonds in a protein constantly vibrate, the higher the temperature, the higher the vibration. Theferore, every once in a while, the protein will, by chance, "jump" to a conformation that it can't come back from. Thermostabile proteins are more stably folded, the vibration is more restricted, so the jump happens less often. There is no ideal temperature to retain conformation; basically the colder the better. That's why proteins always need to be kept on ice when working with them and they need to be stored at -80 degrees C.


I just thought that proteins have evolved to have a particular conformation in a particular environment, according to exactly what their function is and where it occurs in (or out of, I guess) the body. Thus natural selection would have favored the amino acid sequence that allows the protein to stably maintain that conformation in that environment. And that would be its "ideal" environment, whatever that entails (termperature, pH, presence of other proteins/compounds, etc). This is of course assuming that stability is required for the job at hand.

But I see what you're saying. The chemical interactions that maintain a protein's conformation are themselves simply not that stable, and will eventually lose their conformation.
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Re: The Effect of Changing the Concentration of an Enzyme...

Postby jebus197 on April 5th, 2010, 11:28 pm 

I think the question you were asking is can proteins/enzymes continue to operate stably/indefinitely, so long as they remain constrained within certain optimum parameters? (Whatever these parameters might be).

This is similar to my own question, but I'm not sure it has been fully answered yet.

My guess is given the information supplied so far, that the answer is no. There is an optimum functional range for enzymes, as we have seen. However this may (although not always, if the process is reversible) result in enzymes gradually becoming denatured and losing their ability to continue to produce new product. ("They might jump to a shape they can't jump back from.") (In fact this is an inaccurate depiction, since strictly speaking from the protein/enzymes perspective in the long run the process is always likely to be irreversible. This is because in order for the process to continue to occur (if we consider this to exclusively be measured in terms of net product), new enzyme must continually be produced, as old enzyme progressively becomes denatured and is used up and (presumably?) is removed from the body as a waste product. So while the "process" may be reversible, and an enzyme may be able to participate stably in a great many reactions over a given period of time, the outcome for the enzyme itself is always ultimately irreversible.

Even when stored at much lower temperatures, given that I know that meat and other products can't be stored indefinitely, I would assume that this denaturing process for proteins and enzymes would continue to occur. So I wonder how long it is feasible to store enzymes for? Is -80 a temperature at which (regardless of how long they are kept) this denaturing process becomes so slow and so gradual that it can almost be considered negligible?

Is it also fair to say therefore that the type of enzyme typically used in this type of experiment would also continue to decay, even when kept at very low temperatures?

It's not entirely linked I suppose, but I'm also curious concerning how low temperatures affect the formation of peptide bonds in protein dehydration synthesis/condensation reaction? (And also it's mirror opposite, as in the hydrolysis/ reaction that occurs when the peptide chains of proteins break down?) Or in other words, what is the exact mechanism and effect of low temperatures on protein formation? (And while you're at it, if you feel up to it, could you please explain the process of decay under normal temperatures in reference to these reactions? Lol.)

In any case it's an oddity of life perhaps (although it appears to be a true statement) that nothing created by life processes (and nothing that the product of those processes in turn produce) is ever built to last. Everything that in any sense can be considered to be in any way engineered, appears always to have a definite start and an almost equally definite end. (With time alone probably being the only meaningful remaining variable). This seems to be equally as true of all biological processes, as it is for anything that we as humans might produce. (Which is quite a strange thought, lol).
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Re: The Effect of Changing the Concentration of an Enzyme...

Postby BioWizard on April 6th, 2010, 2:38 am 

Obviously in this case, the increased concentration of enzyme results in an increased rate of reaction, up to a given point when all of the active sites of the enzyme are used up.


That statement is phrased weird. The more enzyme you add, the more sites there is. Thus, as you add enzyme, you increase the concentration of available sites, not the other way around. I suppose what you meant to say is "up to a given point where all the substrate molecules are bound to enzyme".

However the question that occurred to me (and one not asked in the essay) is what would happen if you just kept adding more substrate


If the substrate concentration is saturating, you achieve steady-state kinetics.

and simply replenished the substrate as it was used up?


The enzyme will continue to convert substrate into product for as long as it's active.

In other words, would the enzymes continue to work indefinitely? Would they never stop working? And if they did stop working, at what point would this be and why?


Depends on the stability of the enzyme and the conditions that affect it. If you keep the enzyme in a thermodynamically stable range, out of reach of proteases, it might continue to work indefinitely. Of course, that's almost never entirely practical.

P.S: this definitely belongs in Biochemistry.
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Re: The Effect of Changing the Concentration of an Enzyme...

Postby BioWizard on April 6th, 2010, 2:41 am 

jebus197 wrote:It's not entirely linked I suppose, but I'm also curious concerning how low temperatures affect the formation of peptide bonds in protein dehydration synthesis/condensation reaction? (And also it's mirror opposite, as in the hydrolysis/ reaction that occurs when the peptide chains of proteins break down?) Or in other words, what is the exact mechanism and effect of low temperatures on protein formation? (And while you're at it, if you feel up to it, could you please explain the process of decay under normal temperatures in reference to these reactions? Lol.)


This can be a separate topic in Biochemistry.

In any case it's an oddity of life perhaps (although it appears to be a true statement) that nothing created by life processes (and nothing that the product of those processes in turn produce) is ever built to last. Everything that in any sense can be considered to be in any way engineered, appears always to have a definite start and an almost equally definite end. (With time alone probably being the only meaningful remaining variable). This seems to be equally as true of all biological processes, as it is for anything that we as humans might produce. (Which is quite a strange thought, lol).


This can be a separate topic in Biology. Although, I think we've already discussed something similar before, regarding how life components are in fact required to be dynamic and short lived to allow for homeostasis. Try doing a search, and if nothing turns up, feel free to start a new thread on it.
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Re: The Effect of Changing the Concentration of an Enzyme...

Postby jebus197 on April 6th, 2010, 3:45 pm 

This can be a separate topic in Biochemistry.


Thanks I might do that.


This can be a separate topic in Biology. Although, I think we've already discussed something similar before, regarding how life components are in fact required to be dynamic and short lived to allow for homeostasis. Try doing a search, and if nothing turns up, feel free to start a new thread on it.


Thanks again. But this wasn't really a question, just an observation, that basically anything that is created by life is subject to fault and decay. (This includes not only biological process, but everything that is man made/engineered by humans too.) Is was an observation made in the context of enzymes and how even at very low temperatures they are unlikely to last indefinitely.
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Re: The Effect of Changing the Concentration of an Enzyme...

Postby BioWizard on April 6th, 2010, 7:52 pm 

jebus197 wrote:Thanks again. But this wasn't really a question, just an observation, that basically anything that is created by life is subject to fault and decay. (This includes not only biological process, but everything that is man made/engineered by humans too.) Is was an observation made in the context of enzymes and how even at very low temperatures they are unlikely to last indefinitely.


Yep, that's entropy for you. It extends to everything in the physical universe.
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Re: The Effect of Changing the Concentration of an Enzyme...

Postby Natural ChemE on April 8th, 2010, 3:08 am 

jebus197 wrote:Obviously in this case, the increased concentration of enzyme results in an increased rate of reaction, up to a given point when all of the active sites of the enzyme are used up.


You'd still get a boast from increasing enzyme concentration, even if there are already an excess of active sites.

It's kinda like, if you're at Wal-Mart, you can ring your stuff up pretty quickly if there's a clerk who is free. But, if the Wal-Mart had even more free clerks all around the store, one of them'll probably be closer to you, so you can check out faster.

Same deal with the molecules. If they're randomly bouncing around, the more active sites, the faster they can go. You won't really notice the speed increase, though, if the reaction at the active site takes a relatively long time compared to the time it takes for the substrate to get to an active site.

More clerks at Wal-Mart won't help speed up your buying process that much if it only saved you 10 seconds to get to a closer clerk but the clerks all take an hour to check out you. But if clerks ring you up instantly, then having a clerk closer by significantly speeds up your buying process.

jebus197 wrote:In other words, would the enzymes continue to work indefinitely? Would they never stop working? And if they did stop working, at what point would this be and why?


They could, until they break down, if you keep removing the products and adding reactants.

Enzymes do break down though. Your body's constantly making new ones.
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Re: The Effect of Changing the Concentration of an Enzyme...

Postby BioWizard on April 8th, 2010, 9:57 am 

Great post NCE. But just to avoid confusion (in a related thread), it might be worth adding that enzymes break down under the action of other enzymes (proteases and peptidases).
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