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Specialty Chemistry Forums => Biochemistry and Chemical Biology Forum => Topic started by: gonzo on February 27, 2013, 01:25:14 PM

Title: Confused about missing H in glycolysis
Post by: gonzo on February 27, 2013, 01:25:14 PM
Hi everyone,

I have some confusion about some steps in glycolysis where I can't account for the loss and addition of a hydrogen atom. Hopefully you can help.

The first is from the step between glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate (not sure how to type formulas here, so bear with me please, and P is the whole phosphate group)

H
C=O
CHOH
CH2-O-P

->

P-O-C=O
       CHOH
       CH2-O-P

with the payoff of NAD+ -> NADH +H+

Now I count 5 hydrogen atoms in glyceraldehyde-3-phosphate and if 2 of them are transfered to NADH +H+ through oxidization how come there are still 4 hydrogen atoms in 1,3-biphosphoglycerate?

My second issue is in the last step - phosphoenolpyruvate to pyruvate. This time a hydrogen atom is added, but I don't understand how that can happen while producing 2 ATP, where is all this energy coming from in this reaction? I read that PEP is very unstable, but guess I'd like some more information to understand better.

O-
C=O
C-O-P
CH2 (double bonded carbon)

->

O-
C=O
C=O
CH3

payoff 2 ADP -> 2 ATP
Title: Re: Confused about missing H in glycolysis
Post by: Arkcon on February 27, 2013, 03:38:19 PM
Dangit.  Where did that mono-atomic hydrogen go, it was right here a min ... *farts fire*  Ah, there it went.  ;D

OK.  Look at this Wikipedia page here: http://en.wikipedia.org/wiki/Glycolysis#Pay-off_phase (scroll down a bit)  The extra hydrogen comes as a cation for the phosphate group.  You will encounter more problems like these, with unaccounted for reactants, and I'm sorry the biology texts aren't more explicit.  Maybe some are better.  But the environment with in the mitochondria contains all the H+ the reactions will need.
Title: Re: Confused about missing H in glycolysis
Post by: Babcock_Hall on February 28, 2013, 09:40:26 AM
There are a couple of questions here.  The thermodynamic favorability of transferring a phosphoryl group from PEP to ADP is partly a result of the favorable free energy of enolpyruvate becoming pyruvate (a keto-enol equilibrum issue).  I found a citation and posted it here about six months ago.  There are two PEPs produced for every one glucose; therefore, a total of two ATPs are made.

The other thing I would suggest is to make sure that you clearly understand the differences among proton transfer, hydrogen atom transfer, and hydride ion transfer.  IMO following the electrons is often more enlightening about some biochemical processes than following the protons is.
Title: Re: Confused about missing H in glycolysis
Post by: Dan on February 28, 2013, 10:27:29 AM
(not sure how to type formulas here, so bear with me please, and P is the whole phosphate group)

See Formatting Guidelines (http://www.chemicalforums.com/index.php?topic=59314.0).

Title: Re: Confused about missing H in glycolysis
Post by: gonzo on February 28, 2013, 10:29:37 AM
Thank you both.

Yes, I guess my problem is with discerning which transfer it is. I'm looking for electron transfers through this whole process as I'm basically trying to keep score of the energy going towards the electron transport chain. I have the same problem at a couple of points in the citric acid cycle, not being able to explain a couple of hydrogens.

If we take the first example I posted, the wiki article says that the Pi is an HPO4-- that dissociates to give the extra H+ (although I'm also looking for an electron here.. what am I missing?)

I'm using Campbell/Reece's Biology intro book and I like it, but it doesn't go into a lot of detail about how to better follow the electron transfer and it's not the easiest area for me. There's useful info on how energy is transferred in redox reactions and then it proceeds with cellular respiration, but I feel I'm lacking some info to analyze each step better.

To take another example: In the citric acid cycle isocitrate drops a CO2 and then is oxidized to give another NAD+ -> NADH +  H+ payoff. But again I can't account for both electrons:

isocitrate:

     COO-
     CH2
    HC-COO-
HO-CH
     COO-

->

alpha-ketoglutarate:

COO-
CH2
CH2
C=O
COO-

so here the middle COO- being dropped means that as CO2 is lost we are left with the electron from the anion and can pick up an H+ from the enviroment to bind to ketoglutarate I assume? That accounts for the ´3 carbon and that way the other 2 hydrogen are accounted for when oxidized. Is this correct? I'm trying to piece it all together.

Title: Re: Confused about missing H in glycolysis
Post by: Babcock_Hall on February 28, 2013, 10:48:34 AM
Assigning and Using Oxidation Numbers in Biochemistry Lecture Courses,  Journal of Chemical Education 2000;77(11):1428-1432.
http://pubs.acs.org/doi/pdf/10.1021/ed077p1428

Bentley et al., Oxidation Numbers in the Study of Metabolism, BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Vol. 30, No. 5, pp. 288–292, 2002
https://facultystaff.richmond.edu/~jbell2/288.pdf

One way you can account for electrons is to assign oxidation numbers to all carbon atoms.  When the oxidation number becomes more positive, there was a loss of one or more electrons.  One way to assign oxidation numbers is:

ON (C) = (# of bonds to oxygen or nitrogen) - # of bonds to hydrogen).

This is the method suggested in the first of the two references.  The second reference above uses a different formalism, but the two methods provide the same answer for NAD:  NADH is two electrons reduced, relative to NAD (from the transfer of a hydride ion).  I am not sure I understand your question with respect to isocitrate, but why don't you try assigning oxidation numbers and see what happens.  Please let us know what you find.
Title: Re: Confused about missing H in glycolysis
Post by: Babcock_Hall on February 28, 2013, 03:21:12 PM
It looks to be in balance when I assign oxidation numbers to all of the carbon atoms.
Title: Re: Confused about missing H in glycolysis
Post by: Yggdrasil on March 02, 2013, 03:55:28 PM
Thank you both.

Yes, I guess my problem is with discerning which transfer it is. I'm looking for electron transfers through this whole process as I'm basically trying to keep score of the energy going towards the electron transport chain. I have the same problem at a couple of points in the citric acid cycle, not being able to explain a couple of hydrogens.

If we take the first example I posted, the wiki article says that the Pi is an HPO4-- that dissociates to give the extra H+ (although I'm also looking for an electron here.. what am I missing?)

I'm using Campbell/Reece's Biology intro book and I like it, but it doesn't go into a lot of detail about how to better follow the electron transfer and it's not the easiest area for me. There's useful info on how energy is transferred in redox reactions and then it proceeds with cellular respiration, but I feel I'm lacking some info to analyze each step better.

To take another example: In the citric acid cycle isocitrate drops a CO2 and then is oxidized to give another NAD+ -> NADH +  H+ payoff. But again I can't account for both electrons:

isocitrate:

     COO-
     CH2
    HC-COO-
HO-CH
     COO-

->

alpha-ketoglutarate:

COO-
CH2
CH2
C=O
COO-

so here the middle COO- being dropped means that as CO2 is lost we are left with the electron from the anion and can pick up an H+ from the enviroment to bind to ketoglutarate I assume? That accounts for the ´3 carbon and that way the other 2 hydrogen are accounted for when oxidized. Is this correct? I'm trying to piece it all together.

If you've taken organic chemistry, it might be useful to look at the arrow pushing mechanism shown on the wikipedia page for isocitrate dehydrogenase (https://en.wikipedia.org/wiki/Isocitrate_dehydrogenase#Catalytic_mechanism).  Some biochemistry text books (e.g. Voet & Voet) may also have a more detailed discussion of the enzyme's mechanism than a general introductory biology text.
Title: Re: Confused about missing H in glycolysis
Post by: Babcock_Hall on March 04, 2013, 10:32:45 AM
@OP, Note that the hydride transfer takes place before decarboxylation, not after decarboxylation.  How does the timing facilitate the reaction?
Title: Re: Confused about missing H in glycolysis
Post by: gonzo on March 06, 2013, 12:42:09 PM
Thank you everyone for your replies, I've been away a bit with illness, but tried to figure out this oxidation number thing now from the linked pdf. Unfortunately it seems each time I try to count I get different numbers..

I was using the isocitrate -> alpha-ketoglutarate example and my numbers just keep turning out all wrong.. counting oxidation numbers on the carbon I go from +9 to +6 ???
Title: Re: Confused about missing H in glycolysis
Post by: Babcock_Hall on March 06, 2013, 02:11:26 PM
For the five carbon atoms of alpha-ketoglutarate, I get +3, +2, -2, -2, and +3.  We need to also remember that CO2 is produced, and its carbon has an oxidation number of +4.  Why don't you provide the six oxidation numbers for the six carbons of isocitrate?
Title: Re: Confused about missing H in glycolysis
Post by: gonzo on March 07, 2013, 06:51:28 AM
I did forget the CO2, but I still seem to manage new numbers, here's what I get:

Isocitrate:

     COO-
     CH2
    HC-COO-
HO-CH
     COO-

Using electrons gained/lost +n = charge

COO- means 3 electrons lost and the charge is -1 so:
-3 +n = -1
meaning n = +2
and for the subsequent carbons I get -2; -1; +2; 0; +2; in total +3

alpha-ketoglutarate:

COO-
CH2
CH2
C=O
COO-

I get +2; -2; -2; +2; +2; in total +2

So my numbers don't match up with yours and they don't seem to match up with one another either..
Title: Re: Confused about missing H in glycolysis
Post by: Babcock_Hall on March 07, 2013, 10:02:51 AM
I am not sure, but you may be confusing formal charge with oxidation number, or perhaps the issue revolves around which atom has the charge.  None of the carbon atoms is charged in alpha ketoglutarate (two oxygen atoms are).  One way to calculate the oxidation number of carbon is ON (C) = (# of bonds to O, N, etc.) - (# of bonds to H).  This is the formalism suggested by Halkides (2000).  Another way to calculate the oxidation number of carbon is Σ electrons gained (lost) + n = 0.  The letter n represents the oxidation number in the formalism given by Bentley et al, 2002.  For carbon number 2 (the ketone carbon) -2 is the number of electrons lost to oxygen.  -2 + n = 0, and n = 2.  When an atom is charged, the charge replaces the zero in the formula above.
Title: Re: Confused about missing H in glycolysis
Post by: gonzo on March 08, 2013, 11:11:44 AM
The latter approach was the one I was using, I'm not sure what you say I'm doing wrong? Like you said, with the ketone it is -2 + n = 0 so n = 2 which I also wrote it as..

I'm a little confused about how to do the charges, I thought I had to do it for each group, so that in COO- carbon has three bonds to oxygen (-3) and the oxygen has a negative charge, thus I thought it would be -3 +n = -1 so that n = 2.

How do I incorporate the charges of oxygen if not in that way?
Title: Re: Confused about missing H in glycolysis
Post by: Babcock_Hall on March 08, 2013, 11:59:10 AM
With respect to carboxylate groups, according to the formalism in the first paper
ON (C) = 3 - 0 = +3
This paper does not treat oxidation numbers on atoms other than carbons.

I am more familiar with the formalism in the first paper than I am with the formalism of the second, but here goes:
For the carbon atom I would calculate -3 + n = 0; therefore n = +III
For the oxygen atom with a negative charge, I would calculate (+1) + n = -1 ; therefore n = -II
For the oxygen atom w/o a negative charge, I would calculate (+2) + n = 0 ; therefore n = -II

In the three bonds to oxygen, the oxygen owns the electrons.  Also the oxygen atom, not the carbon, has a formal charge of -1.  The Bentley paper gives +III as the oxidation number for Carbon-1 of the acetate ion in Table III.

What do you calculate for isocitrate?
Title: Re: Confused about missing H in glycolysis
Post by: gonzo on March 08, 2013, 12:55:38 PM
Sorry, but I don't get it :(

I thought I was supposed to count the oxidation numbers for carbon (I was looking at the Bentley paper and how it described that all the interesting stuff in NAD+ reduction really took place on the carbons and not the charged nitrogen atom). If I'm also counting for oxygen I get confused because then I don't know how to pick out one from the other - as you say in a carboxyl group the electrons 'belong' to the oxygen. But that's sorted by giving them away as per the formula.

I know that it's the oxygen atom that has the charge, but I still have to include that charge somehow. You're right that it says in table III that carbon has an oxidation number of +3, but shouldn't that be something that can be shown from the formula without having to look it up?

So I don't know if I'm supposed to count for both oxygen and carbon now (and if so, also any other atom in the molecule I suppose). I understood the trick as simply seeing whether carbon 'owned' or didn't own the electrons and then adding charges for each group.

Maybe it's just because I don't really get what I'm counting. From I understand redox reactions the whole point is not just the transfer of electrons but the energy level of said electrons (thereby transfering energy with them), and that depends on the bonds.. thus I'm confused about how one can keep score simply by tallying in this way.

But then again, I can't figure that out anyway.

Ignoring that, isocitrate would then be:

     COO-
     CH2
    HC-COO-
HO-CH
     COO-

+3; -2; -1; +3; +0; +3; making the oxidation number 6

doing the same with alpha-ketoglutarate:

COO-
CH2
CH2
C=O
COO-

+3; -2; -2; +2; +3; making the oxidation number 4, so it still doesn't add up, does it? Considering we also have a CO2 to account for. Ugh, chemistry.

(edited)
Title: Re: Confused about missing H in glycolysis
Post by: Babcock_Hall on March 08, 2013, 01:44:05 PM
The reason I brought up acetate in Table III was to show that the calculation I presented was correct.  With respect to isocitrate, It looks as if you have done the calculation correctly.  So the sum of the oxidation numbers of carbon atoms on the reactant side is +6, and the sum of oxidation numbers of carbon atoms on the product side is +4 + +4 = +8.  Thus, isocitrate is 2 electrons more reduced than alpha-ketoglutarate plus carbon dioxide.  Those are the two electrons picked up when NAD becomes NADH.  Does this help?
Title: Re: Confused about missing H in glycolysis
Post by: gonzo on March 08, 2013, 03:00:41 PM
Yes, it helps sort of, but I'm confused about how to properly calculate charged segments like COO- if I can't find them in a table.. I understand that it works out to +3 for the carbon atom, but what about the charge? Even though it's oxygen that's charged, the charge still matters, doesn't it?

I wasn't implying that your calculation was wrong, I'm just confused about chemistry. And I still don't understand the deeper concept here because just tabulating electrons can't be the whole story given that their energy level varies depending on the bond..

I also have another question about an oxygen popping up in the citric acid cycle, so it's along the same lines, can I use the same thread?
Title: Re: Confused about missing H in glycolysis
Post by: Babcock_Hall on March 08, 2013, 04:41:04 PM
With respect to the carboxylate group, I think you should treat each atom individually, not as a segment.  Also, if you do the calculation by the method of the 2000 paper or the 2002 paper, the oxidation number of the carbon atom comes out the same, which should be reassuring.

I think tabulating oxidation numbers can be helpful in biochemical processes for a number of reasons.  It can allow one to check whether any proposed anaerobic catabolism can actually happen.  For example, glucose can be catabolized to two molecules of lactic acid, and one can check the oxidation numbers to see that they add up to the same value on reactant and product side.  Aerobic catabolism does allow for the sum of the redox numbers of the carbon atoms of metabolites to be different, but the oxidation numbers of the carbon atoms in NAD or FAD should change accordingly.

Another thing is that some reactions may not look like redox processes, until you check the oxidation numbers.  Aminotransferases are a good example; glutamate synthase and thymidylate synthase are two more.  It is difficult to say whether or not your question belongs in this thread, but I see no reason not to post it here.
Title: Re: Confused about missing H in glycolysis
Post by: gonzo on March 09, 2013, 02:28:15 PM
Hmm, if I treat each atom individually does it go like this?

COO- is +3 for carbon, -2 for double bound oxygen and -2 for negatively charged oxygen (1 +n = -1 so n = -2) giving a combined oxidation number of -1 for the caroboxylate group?

if I do that for all oxygens in isocitrate I get -14 and for hydrogen +5, combined with the +6 for carbon that's n=-3

doing the same for alpha-ketoglutarate I get -10 for oxygen, +4 for hydrogen and the +4 for carbon makes n=-2

for the CO2 molecule it cancels out to 0.

So that didn't work or I made mistakes (likely).

I couldn't read the 2000 paper that you linked to, I have to be a member or pay for it, that's why I've been using the Bentley method since it was free.

I do understand that oxidation numbers can be useful for the reasons you give, I just don't really understand how they work because there must be more to it, energy levels depend on electron orbits and here the only deciding factor for electrons being counted one way or the other seems to be which atom is more electronegative. I can't see how that's the whole picture, but if it works it works I guess.

Also, thanks for all the help, even if I am confused about a lot of stuff it helps to juggle it around a bit.
Title: Re: Confused about missing H in glycolysis
Post by: Babcock_Hall on March 11, 2013, 09:46:05 AM
I do understand that oxidation numbers can be useful for the reasons you give, I just don't really understand how they work because there must be more to it, energy levels depend on electron orbits and here the only deciding factor for electrons being counted one way or the other seems to be which atom is more electronegative. I can't see how that's the whole picture, but if it works it works I guess.

Also, thanks for all the help, even if I am confused about a lot of stuff it helps to juggle it around a bit.
The question of energy in biochemical redox reactions is a separate matter, but I can give you a thumbnail sketch.  The oxidation of NADH to NAD by molecular oxygen is thermodynamically quite favorable.  The electron transport chain carries out this oxidation in many small steps, with the creation of a protonmotive force (basically protons are moved uphill in an electrochemical sense).  When these protons move back downhill, this movement is coupled to the synthesis of ATP.  I will have to leave the rest of your post for later.
Title: Re: Confused about missing H in glycolysis
Post by: gonzo on March 11, 2013, 11:36:42 AM
Yes I understand this part fine, it's more about the concept of oxidation numbers as simple integers that can be tabulated in this way, it seems to me it must be a whole lot more complicated than that :)
Title: Re: Confused about missing H in glycolysis
Post by: Babcock_Hall on March 11, 2013, 06:46:59 PM
I have gotten busy at work and may not be able to write a lengthy response for a day or two.  Can you obtain the 2000 paper from your library via interlibrary loan?  It is simpler, but perhaps less general, than the 2002 paper.  With respect to your question, the integer nature of oxidation numbers seems inevitable when one assigns ownership to one or to the other atom completely.
Title: Re: Confused about missing H in glycolysis
Post by: gonzo on March 12, 2013, 04:39:37 PM
First of all, thanks again for all the help. I don't think I can get that paper from the library at the moment, but I'm ok with using the other method.

I understand why they have to be integers the way oxidation counting works, my question is more in terms of why it works on the physics level. I'd think that, say, hydrogen electrons bonded with oxygen would have a different energy level than carbon electrons. But that doesn't seem to be reflected when counting oxidation numbers (oxygen 'owns' them in both cases), so I'm just not sure how the true energy changes are accounted for. Not sure I'm explaining myself very well, but even from a simple electron shell perspective it seems weird to me. I'm not saying it's wrong (I remember it from chemistry classes), I'm just not really clear on why it works.
Title: Re: Confused about missing H in glycolysis
Post by: Babcock_Hall on March 15, 2013, 09:24:57 AM
The sum of all of the oxidation numbers of each atom in a chemical species is equal to its charge, and that is what you have shown above.  If you want to balance charges in this reaction you have to include the conversion of NAD to NADH.  If I were going to do that as an exercis, I would replace the large adenosine diphosphoribosyl (ADPR) group with something smaller, like a methyl group.  Those atoms don't change oxidation number, so a little bit of simplification is justified in my opinion.

If you just look at the sum of oxidation numbers on carbon atoms, you will see that for isocitrate they sum to +6; for alpha-KG, they sum to +4; and for carbon dioxide, it is +4.  Looking at it just from the point of view of carbon atoms allows one to see that the carbons have become oxidized by two electrons, which is balanced by the reduction of NAD by two electrons.
Title: Re: Confused about missing H in glycolysis
Post by: gonzo on March 19, 2013, 10:19:32 AM
I thought that NAD+ simply obtained two charges, so I didn't think it would be necessary to count them if the difference was already known to be two..

About the added oxygen in the citric acid cycle I wanted to ask earlier; the diagram in my book doesn't show how it's added when acetyl CoA reacts with oxaloacetate, but in this wiki a hydration reaction is shown http://en.wikipedia.org/wiki/File:Citric_acid_cycle_with_aconitate_2.svg - I still can't see from that either how the oxygen is added between succinyl CoA and succinate though. I see the substrate level phosphorylation taking place, but where does the oxygen come from, is that just a hydration reaction not shown?
Title: Re: Confused about missing H in glycolysis
Post by: Babcock_Hall on March 19, 2013, 10:57:24 AM
I just have time for a quick response today.  We have to account for the two electrons lost by carbons in the isocitrate-to-alphaKG+CO2 conversion.  Those two electrons reduce (change the oxidation number of) two carbons in the nicotinamide ring of NAD.  I think that the 2002 paper does a nice job going through NAD versus NADH in this regard.

Molecular oxygen is not part of the citric acid cycle, but I think that there is one molecule of water in the citrate synthase reaction.  One has to convert citryl CoA to citrate.  When one makes GTP from GDP and inorganic phosphate, one molecule of water is produced.  I believe that this accounts for the oxygen you are thinking of in comparing succinyl CoA to succinate.  This reaction trades one "high energy" bond for another. 
Title: Re: Confused about missing H in glycolysis
Post by: gonzo on March 20, 2013, 04:43:35 PM
Yes that would account for the extra oxygen between succinyl CoA and succinate if there's a water produced from the reaction (I can't seem to find details of this anywhere, but I'm curious about it).
Title: Re: Confused about missing H in glycolysis
Post by: Babcock_Hall on March 20, 2013, 04:51:06 PM
The combination of phosphate and GDP is basically the reaction of two acids forming an anhydride bond.  That produces water.  Conversely, if we hydrolyzed GTP, our products would be GDP and phosphate.