1. In class you said that 8 salt linkages break when O2 is bound to heme. Are the salt bridges the 5th
coordination?

No, you have the wrong picture for where the salt linkages are. Read Stryer Chapter 7 to find the
exact location of these salt linkages, which are either between the alpha and beta subunit globins or within them, not to the heme. The salt linkages stabilize the deoxyhemoglobin (T) structure.

If so, wouldn't only four be broken in hemoglobin since there are only four 5th coordination bonds?

See above

2. When at rest, do your muscles access O2 from the myoglobin 1st, and then if that runs out, it
accesses O2 from hemoglobin, or is it vice versa? Does this same order of O2 access apply when your muscles are exhausted after a workout (Mb then Hb4, or Hb4 and then Mb as back up)???

Myoglobin is like a "sink" or storage container. Oxygen transport to the muscle occurs
constantly, but demand will be highest when the muscle is active. Hemoglobin brings oxygen to the muscle where myoglobin stores it. In active exercise the muscle cells will draw both from the myoglobin and from the hemoglobin.

3. Re: assignment question: E7His function #2: When you said H bonds to O2, increasing O2 binding,
what did you mean? What H bonds are you referring to. I understand that E7 disables CO from binding linearly, and lets O2 bind linearly, but didn't understand the H bond comment in class.

In class I showed what is not shown in Stryer, namely that the oxygen bound to the heme is also
bound to the E7 H atom on the histidine -NH group. This sharing of the H atom between the N and the oxygen is a hydrogen bond. It increases the affinity of the binding of heme to oxygen.

Also, is E7 bound to Fe when there is no CO or O2 bound to the ion?

E7 is never bound to Fe(II), only the F8 His is bound to Fe(II).

Q.2. Hi Prof Mellors,

I have a question regarding hemoglobin. In the text book at pg 160, it
says, "...in the absence of BPG, the P50 of Hb is 1 torr, like that of
myoglobin."

I understand, without BPG, Hb and Mb have the same affinity for oxygen. But
won't the cooperativity of Hb result in a higher oxygen saturation? In other
word, would cooperativity increase the affinity?

Thank you very much. A.

You are quite right about what you have read. The loss of BPG abolishes co-operativity because co-operativity depends on a balance between low affinity T forms and high affinity R forms. When BPG is not present in the RBC, only the R form is found and it has no capacity to interconvert to the T form since the latter is stabilized by BPG.

Hope this helps,
Alan Mellors

Q.3.

C wrote:

Dr. Mellors:
I noticed that in our course handout there is an article of some sort regarding carbon monoxide and I was wondering if we are responsible for this info for the final?
Thank you.
C

Only the information on CO that we used in the lecture is to be tested on the exam.
Alan Mellors

Q.4.

J wrote:

Hi Dr. Mellors,
re: chem 3560
I am confused about why cyanide is poisonous and why nitrite is used as an antidote for cyanide poisoning. This is question 2 from assignment #3. I know we went over it in lecture,
but it is still unclear to me. If you could explain it to me that would be great.
Thank-you,
J.

Cyanide anion CN- has a affinity for FeIII or Fe3+. It poisons respiration by combining with the Fe3+ of cytochromes at the end of the electron transport chain where oxygen is reduced to water. If nitrite is given to the patient, it converts some of the RBC hemoglobin (heme Fe II) to methemoglobin (heme Fe III). This soaks up the CN- from the blood and prevents it reaching the respiring tissues.

Alan Mellors

Q.5.
W wrote:

Dear Dr. Mellors I have two conflicting things in my notes For the Hill plot, I have that n cannot be greater then 4 for O2 binding, and that for Hb4 A it is 2.8, but then I have n=10 for aggregation of deoxy
HbS. If n cannot be greater then 4, how is n=10.
W.

Dear W,
If binding was infinitely co-operative (and it never is) then the Hill plot n would equal the number of binding sites. How many binding sites do you think there are for deoxyHbS?

There will be a very large number of binding sites as the fibres come together to form cables, much greater than 10 binding sites, so the large n indicates a high degree of coop for many sites.
Alan Mellors

Q. 6.
R wrote:

Pr.Mellors,

Sickle Cell Anemia when diagnosed in utero & the procedure of restriction endonuclease digestion of the DNA sample which leads to southern blotting.

I have read page 173 in the text & looked over the notes, yet the 1.1 - 1.3kb fragment labelling via the probe etc I do not understand.

Can you pleas explain the procedure of Southern blotting. Any information would be greatly appreciated.
Thanking You,
Sincerely
R

R,
Look at the web demonstration at http://vector.cshl.org/Shockwave/southan.html

The only difference is that the SCD test uses a probe specific for the beta globin gene, and detects when the gene is cut to a 1.1 kbp piece (normal beta) or when the mutation is present and the piece is not cut at the codon specifying Val instead of Glu so cleavage yields a longer piece 1.3 kbp.
Get back to me if you have more questions,
Alan Mellors

Q. 7.
A wrote:
Dr. Mellors,
I'm in your Biochem. II class and I'm studying the Pentose Phosphate Pathway right now. I was just wondering how much of it do we have to know for the final. i.e. oxidative, non-oxidative, structures, names of enzymes, names of intermediates.
Thank you,
sincerely,
A

Dear A,
You should know the names, structures and cofactors for the first three enzyme reactions. You should know the balanced equations for six glucose 6 phosphates going through PPP and reverse glycolysis to give 5 fructose 6 phosphates. You should know the experimental tests for measuring PPP compared with glycolysis.
Alan Mellors

Q. 8.

Professor Mellors,
The following material does not appear in the lecture note material but are described in detail in the textbook readings. How responsible are we for:

1) Pg 596-597: Protein phosphatase 1 - how it reverses regulatory effects of kinases on glycogen metabolism.

Just know that it does.

2) How insulin stimulates glycogen synthesis by activating protein phosphatase 1?

Again, just know that it does.

3) Glucose activation of glycogen synthase, glycogen storage diseases and glycogenin (587).

Not needed.

Is glycogen degradation the focus or are we just as responsible for its synthesis?

You need to know the compounds and structures involved in both glycogen synthesis and degradation, and the control of glycogen phosphorylase and glycogen synthase.

4) Pg 561-562: Are we responsible for transaldolase and transketolase reactions of the non oxidative steps in PPP.

No.

5) Pg 563-564: Should we know the four modes of PPP in detail as outlined in the textbook reading.

No.

6) Pg 584: The phosphoglucomutase conversion of glucose1P to glc6P, again should we know this reaction in detail?

Not in detail, just know the substrate and product structure and name.

Finally

7) IN regards to the fatty acid synthesis pathway are we responsible for all the structural formulae and names of the compounds?

You need to be able to draw the structures only, with cofactors named.. You need to know that the enzymes are organized on a homodimer, and to know how the subsequent turns differ from the first turn.

Q.9.

Dr. Mellors:
Could you tell me what the difference is between +homotropic, -homotropic, +heterotropic, and -heterotropic modulators/cooperativity?

If the substrate or transported ligand causes a change in the affinity of the protein for the substrate or ligand, then the effect is homotropic (affecting self). If some other molecule causes altered affinity then it is heterotropic (affecting some other). If the effect is to increase affinity, and therefore activity, it is positive, if decreasing, then it is negative.

Also, how do you tell if there is a defect in the extrinsic pathway as opposed to the intrinsic pathway? And, why are they called that?

The intrinsic pathway means the clotting that occurs in the test tube, out of the body. All the components required are present in the blood sample, that is they are intrinsic to the sample. Clotting that occurs in the body, is faster, because tissue factors not present in blood samples, speed up the
clotting. These factors are extrinsic, that is, outside the blood sample. If clotting is slow relative to normal, in a test tube, there is a defect in the intrinsic or contact pathway. If clotting in the test tube is normal, but slow in the body, then the defect is in the extrinsic or tissue factor pathway.

Q.10.
Hello Dr. Mellors, I was reading about the Pentose Phosphate Pathway in the textbook....and there is quite a bit of detail in the text regarding xylulose 5 -phosphate and sedoheptulose 7 phosphate, as well as three reactions which connect the ppp and glycolysis. I was wondering how much, if any, we had to
know about these things!?
thank you. H

You need to know only the oxidative phase, with structures, and the balanced equations for six
glucose 6 phosphates going by PPP and reverse glycolysis to 5 fructose 6 Phosphates.
Alan Mellors

Q.11.

Hi Dr. Mellors!I
have a question regarding the final for 3560. Do you want us to know the pathway (i.e.
structures, enzs, reactants) of ribulose-5-P to Fru6P via transketolase and transaldolase?

No

Or should we just know the "gist" of it? i.e.. 6 Ru-5-P to 2 3-P-Glyceraldehyde and 4 Fru6P?

Yes

One more question, on the PPP balance sheet you have 2 3-P-Glyceraldehyde being converted to 1
Dihydroxyacetone-P. Why is it only converted to 1 Dihydroxy and not 2? I thought Dihydroxyacetone-P was an isomer of 3-P-Glyceraldehyde?

BECAUSE IF ONE GOES TO DHAP AND ONE STAYS AS 3PG, THEN BOTH CAN REACT
TOGETHER IN REVERSE GLYCOLYSIS TO GIVE FRU 1,6 BIS P.

Q.12
P wrote:

I just have a quick question regarding PFK in control of Glycolysis: Q. If the trigger for H+ inhibition over PFK is a drop in blood pH, this means that lactate wont form excessively. Does this small amt of Lactate raise the blood pH?
P,

The production of lactate and its transport in the blood will lower the blood pH, because a H+ will be exported with the lactate.
Alan Mellors

Q. 12
A wrote:

I just have a question...
I'm finishing up the readings and I'm running into some material that I don't recall being discussed in class. Would something that was not discussed in class be important for me to learn as potential exam material? Or if it was covered could you point out where, I've gone through my notes and can't find anything.

For example - pg. 271 lipid (liposomes) and planar bilayer
- pg. 272 "Transport antibiotics are carriers or channels"
Gramicidin A, valinomycin

A,
Those topics are not included in the exam material or in the lecture material, so are not needed.
Alan Mellors

Q.13
J wrote:

I have a question about action potentials. The way I understand it, a ligand such as acetylcholine first activates a ligand-gated channel, allowing Na ions in. This then changes the membrane potential, which allows the voltage-gated Na channels to open and let Na into the cell, thus the action potential is propagated along the axon. At the peak of the action potential, the Na channels close and the K channels open, repolarizing the membrane by letting K ions out of the cell.

Is the Na/K ATPase involved here? I know it creates the membrane potential in the first place, but I'm confused as to how it is involved in repolarizing the membrane.
J,

The Na+/K+ ATPase is essential for creation of the membrane potential and ion gradients on which the action potential depends. It does not function directly in the events of the action potential. However, a question asking you to name the membrane proteins and events leading to the action potential, would require a description of the ATPase and of the ion channels.
Alan Mellors

Q. 14
Hello Dr. Mellors

I have two questions:

1) what is the significance of the 14 -C in the tests for PPP? I understand the C-1, vs. C-6 part but I don't understand the 14?

14C is a radioactive isotope of carbon 12. If you label glucose at the 1 or 6 position with this isotope then the radioactivity in the CO2 produced will tell you how much oxidation occurred at the 1 carbon compared with the 6 carbon. More radioactive 14CO2 comes from the 1 position than from the 6 position if the PPP is the pathway.

2 ) In Fatty Acid Synthesis are we to learn it as on page 617 (Stryer 4th) or the way you described it in the handout? I am a little confused because the reactions are the same but the text gives them different names, such as the end product in the text being Butyryl-ACP, whereas the note pack have Acyl-ACP as the end four carbon fatty acid. Does it matter which one we learn?

The names in Stryer only apply to the first turn, there are different names for the second turn compounds. I gave you generic names applicable to all turns. However, I will not ask you for names of compounds, only structures, so it does not matter which you learn, if any.

Q. 15
V wrote:

For gluconeogenesis are we responsible for all the enzymes and substrates in the conversion of pyruvate to glucose? In the text book readings these steps are discussed in great detail concerning pyruvate carboxylase and the conversion of oxaloacetate to transport from the mitochondria into the cytosol. Are we also responsible for the structure of pyruvate carboxylase concerning biotin? Or should we be concerned only with the regulation of gluconeogenesis via the bifunctional enzyme?

The only detailed part of gluconeogenesis that we cover in this course is the fructose 1,6 bis phosphate to fructose 6 phosphate enzyme step, and the rest of the structures to make glucose in the liver, namely glucose 6 phosphate to glucose. Nothing else. You should know that the pathway involves both
mitochondrial enzymes and cytosolic enzymes. The details of the enzyme mechanisms e.g. acetyl CoA carboxylase, are not needed.

Hope this helps,
Alan Mellors

Q. 16.
C wrote:

Hi Dr. Mellors, I was wondering if we were responsible for the biotin rxn, or just what was in our lecture notes and also if we were responsible for the steps in GNG taken to bypass the irreversible ones in glycolysis. Also, there are some enzymes in the text not discussed in lectures. Are the majority of your questions based on your lecture notes or from the text book.
Thanks for your help, Cara.

All exam questions will be on material that we have covered in class.
Alan Mellors

Q. 17.
Dr. Mellors,
I have some more questions... sorry to bother you. On the required structures list is says we have to know glucose and fructose and their phosphates --- which phosphates do those include?

Glc1P, Glc6P, Fru1P, Fru6P, and the 1,6 and 2,6 bisphosphates.

Also, do we need to know how to draw turns 1 and 2 of Fatty Acid Synthesis or just the first turn?

FAS turns 1 and 2.

Should we memorize glycolysis, the TCA cycle, beta > oxidation like we had to for CHEM*2580?

No, for the memorization.

And finally, on page 312 of our > text book it talks about E1 and E2 should we worry about that?

E1 and E2 are simply the two conformations of the pump and so whether you call
them that, or draw a cycle, is up to you.

Thank you very much,
A

Q.18.

In the glycolysis pathway, do we need to know all the names, structures, and enzymes from glucose to pyruvate?

I am writing in regards to the final exam. I am wondering how much of glycolysis we are required to know. Are we responsible for the first half only or the entire process? If so, are we required to know all of
the enzymes and their structures?

No, but from glucose to glycogen, and back, and from glucose to fructose 1,6 bis phosphate. If I did not use the name of the enzyme in my lecture, then it is not required, for example, we did not talk about hexokinase or glucokinase which turn glucose into glucose 6 phosphate. We did not talk much about phosphoglucomutase which interconverts glucose 6 P and glucose 1 P.

Q. 19.

I have a few questions about the material for the exam:

1) Are the reactants to produce 1 palmitate: 7 acetyl CoA, 7 malonyl CoA, and 14 NADPH?

No, eight acetyl CoA are needed, seven go via acetylCoA carboxylase to make seven malonyl CoA. So the FAS uses one acetyl CoA and 7 malonyl CoA.

2) Are we responsible for bacteria and lactose and glucose transport (Page 317 in the Fourth Edition)?

No.

3) Are we responsible for the details of the acetylcholine channel (Page 295-297 in Fourth Edition)?

No, just know that it is a ligand gated channel that opens when the ligand acetylcholine binds and it permits the entry of Na+ into nerve cells at the synapse.

4) In the glycolysis pathway, do we need to know all the names, structures, and enzymes from glucose to pyruvate?

No, but from glucose to glycogen, and back, and from glucose to fructose 1,6 bis phosphate.

5) Do we have to know about the effects of protein phosphatase 1 on glycogen?

Just know that it will reverse the effects of PKA in glycogenolysis and glycogen synthesis.

Q.20.
Professor Mellors:
I know that for the mid-term you had said that we would only be required to know one of the tests (our choice) that proved that ATCase was under concerted symmetric control. Is this also true for the final?

Yes

Also, could you tell me what an ester bond is?

An ester bond is made by eliminating water between an alcohol and an acid. Therefore so called anhydride bonds between phosphate and sugar alcohols are esters. So are the bonds between fatty acid carboxylic acids RCOOH and alcohols like glycerol, or thiols like CoA-SH. In each case, water is
eliminated to make the bond, and water is added (hydrolysis) to cleave the ester bond.

Lastly, calcium is involved when prothrombin goes to thrombin, but where is the calcium coming from? Is it just on the platelet membrane? What does phosphatidyl serine have to do with it?

Ca++ is high in the plasma, low in the cytosol. When platelets are activated, the negatively charged phospholipid phosphatidylserine moves to the plasma membrane. This phospholipid is also found on the inner surface of the plasma membrane, so is exposed when tissue cells are broken as in a wound. Now Ca++ will bind to the exposed negative PS, the clotting factors prothrombin, Xa and Va will come together on the membrane and prothrombin cleavage to thrombin will occur at the wound site.
Alan Mellors

Q.21.
I just have a few questions for you regarding some material from Chem 3560; Is phosphorylase phosphatase the same as protein phosphatase 1?

Yes.

Also, when we draw structures such as cAMP are we required to draw the adenine base as well

No

or can we simply just write adenine and show where it would attach to the molecule?

Yes.