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15 Cards in this Set
- Front
- Back
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How does Ribonucleotide Reductase work?
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-takes OH in ribonucleotides (in a nucleoside diphosphate), plucks it out, as OH-, put a proton to it and convert it to water
-then it takes a reducing power, ultimately from NADPH, and attaches H at the T position. This is what makes it a 2’ deoxynucleotide |
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In the process of converting NDP to dNDP, the ribonucleotide is oxidized.
How is this accomplished? How do you regenerate the active form of the enzyme for the following round of catalysis? |
-in the process of catalyzing this reaction, ribonucleotide is going to get oxidized
-there are two cysteine residues that provide the pair of electrons necessary for this reaction -when cysteine residues are oxidized, they become a harmless disulfide bond -the only consequence of that is that we need to reduce the cysteines to their reduced state/the active form for the next round of catalysis -to do that, we need to use GLUTAREDOXIN or THIOREDOXIN, which also has a pair of cysteine residues -->when it gives up its electrons, it forms a disulfide bond and converts ribonucleotide reductase to its active state. -something needs to provide e- to glutaredoxin and thioredoxin to regenerate them to their active, reduced state -we don’t need to go into too many details about that, but know that the reducing potential to reduce ribonucleotide reductase ultimately comes from NADPH |
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Where does the reducing potential to reduce ribonucleotide reductase ultimately come from?
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NADPH
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How is ribonucleotide reductase regulated?
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-There is a dimer of this enzyme
-The dimer has TWO active sites -The dimer has TWO (x2 = 4 total) regulatory sites where nucleotides bind: 1. a 1’ regulatory site -The 1’ site is like an on/off switch -when the ribonucleotide ATP is bound to the 1’ site, the enzyme is ON -when dATP is bound it’s OFF -this is STRICTLY at the 1’ site 2. a 2’ regulatory site -all different nucleotides can be bound at the 2’ site, and different nucleotides have priority in terms of which gets converted and which nucleotide gets converted to deoxynucleotides |
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How does regulation of ribonucleotide reductase differ at the 1' and 2' site?
Why does it have regulation like this? |
1’ Site:
-regulation from right to left -the regulation when ATP or dATP is bound at the primary site so it simply says that when dATP is bound, enzyme is off, when ATP is bound, enzyme is ON (green) it’s DIFFERENT on the 2’ site: -Essentially what happens is that each nucleotide will eventually shut down its OWN synthesis at the substrate specificity site and then promote the next nucleotide to be converted into its own respective dNDP -NO deoxynucleotides present: -you never really have a situation in a cell where there are NO deoxyribonucleotides present at all, but let’s just begin with that particular premise. -at the beginning, all you have present in the cell is ATP, no dATP -you activate the conversion of the PYRIMIDINE nucleotides and give it preference for conversion into their respective deoxynucleotide diphosphates -eg. when only ATP is present, CDP and UDP will be converted to dCDP and dUDP. They will eventually be converted to dCTP and dTTP (red) -once dTTP is formed to high enough levels, it does two things: 1. Shuts down its own synthesis --> shuts down conversion of CDP and UDP to dCDP and dUDP (blue arrow/circle) 2. Activates the conversion of GDP to dGDP -->that will eventually be converted to dGTP -when dGTP accumulates to high enough levels, it also does two things: 1. Shuts down ITS own synthesis 2. Activates the conversion of ADP to dADP and to dATP -when enough dATP accumulates, it binds to the PRIMARY SITE and shuts the entire enzyme off *dATP is given to be an indication that the levels of ALL the dNTPs are present at high/adequate levels Why does it have this weird regulation? -it is the ADENYLATE nucleotides present at high levels, the G are present at the next highest level, the pyrimidine nucleotides are present at the next lowest levels -when RNA polymerases does RNA synthesis, it doesn’t matter if ATP is extremely high and UTP is extremely low. RNA polymerase can handle that readily. DNA polymerase, on the other hand, cannot because it wants more uniform level of the 4 canonical dNTPs |
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Why does DNA polymerase want more uniform levels of the 4 canonical dNTPs?
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-Imagine there is a G residue in the DNA template and the DNA polymerase needs to promote the incorporation of a C residue opposite to that site, so by base pairing the incoming dCTP with a G reside in the DNA template, it’s going to promote the polymerization of the nucleotide at that site
-Now in the language of Jessica Jones (direct quote I kid you not) what that means in terms of enzymatic regulation, the Km for the right nucleotide being incorporated is going to be very very low and the Vmax is going to be very very high -This does NOT mean that the wrong nucleotide (say a dTTP) cannot be incorporated at that site, it only means that the Km is going to be extremely HIGH and the Vmax extremely LOW. -If you dTTP level is extraordinarily high and present at a much higher level than dCTP, then the chances that dTTP will be misincorporated opposite a G residue will become higher and higher, something the cell cannot afford if it wants to copy its genetic material with fidelity and avoid the accumulation of mutations -Thus, DNA polymerase wants uniform levels of all 4 dNTPs |
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Why is the regulation of ribonucleotide reductase by dNTPs so complex?
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-If we were to proportionally convert all 4 of the ribonucleotides to their respective deoxynucleotides, we’re going to have unbalanced levels of all the deoxynucleotides:
-->dATP would be present at the highest concentration wherease dCTP and dTTP would be present at the lowest concentration -->this regulation ensures that the pyrimidine nucleotides, the nucleotides present at the lowest concentration, get first priority to their respective dCTP and dTTP forms -->dGDP gets the next priority -->dADP gets the lowest priority -This does NOT mean that its happening necessarily in this order, it just means that it’s trying to balance the level of synthesis from unbalanced levels of reactants -That is the REASON for the complex regulation |
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How do we synthesize dTMP?
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-synthesis of dTMP molecule -when we UDP and convert it to dUDP, and there is going to a nucleoside diphosphate kinase that will convert the dUDP to dUTP
-a simple deamination of dCTP will convert the dCTP to dUTP -dUTP is not used for DNA synthesis because we have dTTP instead --> thymine residues in DNA, not uracil -there is a very potent dUTPase (think of it as a sanitizing enzyme) that destroys the dUTP as soon as it’s synthesized and it cleaves a PYROPHOSPHATE of the dUTP and converts it to dUMP. -dUMP is methylated to form dTMP by an enzyme called thymidylate synthase -Thymidylate synthase is often a target for chemotherapeutic agents! |
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Describe the Thymidylate Synthase reaction
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The Thymidylate Synthase reaction is as follows:
1. A methyl group is added to dUMP at the 5 position of the thymidine base (green arrow) -thymidylate synthase uses a cofactor known as N5, N10-Methylene-Tetrahydrofolate - ->here is another role of FOLIC ACID as a vitamin leading to an important cofactor in DNA synthesis -here is this methylene group (red) attached to N5 and N10 as shown by circle -what happens is that this methylene group is going to be transferred to the green arrow position, but we also need to REDUCE the methylene group, so we need the hydrogen/electron (pink arrow) to reduce that methylene group to make the methyl group at the 5 position of the thymidine base -there are two things that then happen to tetrahydrofolate: 1. It gives up its single carbon necessary for the 1 carbon transfer that it specializes in. 2. Additionally, the tetrahydrofolate will be oxidized to dihydrofolate -These are the two reactions that take place, but it’s really the dihydrofolate reductase reaction (orange) that is important medically because that can be the target for chemotherapeutic agents --> here is 7,8-Dihydrofolate. We’re going to use NADPH to reduce it back to Tetrahydrofolate and that is catalyzed by DIHYDROFOLATE REDUCTASE -Then serine is going to give up its side chain carbon, and that carbon is used to reintroduce a methylene group at the position indicated by the purple arrow and regenrate N5, N10 – methylene- tetrahydrofolate -To regenerate the N5,N10-methylene-tetrahydrofolate for another round of catalysis, we need to do two things: 1. We need to reduce 7,8-dihydrofolate back to tetrahydrofolate 2. We need to add back the carbon (which can come from SERINE residues) |
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How are purines degraded?
What is this process important for? |
PURINE DEGRADATION
-the end product of catabolism of purine nucleotides is URIC ACID -It can take place in all cells, but it is produced primarily in the LIVER, and this is where it can result in some physiological problems -The liver releases hypoxanthine into the blood stream where other somatic cells (extrahepatic tissue) collect hypoxanthine to make their own purine nucleotides using resalvaging enzymes. **This process is not only important for getting rid of excess AMP and preparing it for disposal/excretion, it is also important for providing the purine base that the extrahepatic tissues will use for their own energy metabolism. There is a DUAL PURPOSE FOR THIS PATHWAY.** __ AMP: Say we have lots of AMP and we don’t need it and we want to degrade it. 1. Dephosphorylation of the AMP nucleoTIDE and it becomes a nucleoSIDE (adenosine) - It’s the dephosphorylation of the nucleotide to nucleoside that is typically the first step in the catabolism of a nucleotide 2. Usually what happens then (it happens with guanine anyway) is the hydrolysis that severs the base away from the ribose moiety. -For some peculiar reason, for adenosine this doesn’t happen until adenosine is deaminated to INOSINE. -->What that means is the base involved here is adenine, that has to be deaminated to hyoxanthine before we can now continue on with the degradation process. ENZYME: adenosine deaminase -this is related to the disease SCID ** remember this enzyme 3. Once we make inosine, we can hydrolyze the base away from the ribose moiety. Ribose is liberated and can return to energy metabolism and we get HYPOXANTHINE -If it doesn’t want the hypoxanthine to be around anymore (say if there’s too much around), some of that will be converted to uric acid. ENZYME: xanthine oxidase -takes H2O and oxidized hypoxanthine and in the process produces hydrogen peroxide which has to be eliminated by scavagening enzymes so it doesn’t cause damage to the cells. -the first step converts it to xanthine --> another step catalyzed by xanthine oxidase converts it to uric acid. This is what is collected by the kidney for excretion __ GMP: A similar process is involved in the catabolism of GMP but this is much simpler than AMP 1. Dephosphorylate the GMP into the nucleoSide guanosine 2. Sever the guanine base from the ribose moiety, giving us Guanine -the liver does not make as much guanine as hypoxanthine, but guanine could be released by the liver into the blood stream for the use of the guanine by other tissues 3. If you want the guanine to be disposed of in the form of urea, we have the enzyme guanine deaminase so we deaminate the guanine after severing the guanine from the ribose (unlike in the case of adenosine deaminase). This produces Xanthine. 4. Xanthine oxidase converts xanthine to uric acid. |
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What's the problem with producing a lot of uric acid???
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-Here is the problem with the production of uric acid
-this is depicted in the ENOL form, and the ENOL form has a pKA of 5.7 -at a physiologic pH (blood pH = 7.4) this is going to be NEGATIVELY CHARGED, so the proton is dissociated from it and it is referred to as a URATE ANION -The urate anion (like sodium urate) has very low solubility in aqueous solution -If sodium urate accumulates to very high levels within areas like the joints, it can crystalize, form little salt crystals in areas of poor circulation. If you form crystals, polymorponuclear leukocytes (PMNs) – essentially phagocytizing white blood cells – can take up that crystal and phagocytize those sodium urate crystals. -In response to taking these crystals up, the white blood cells release hydrolytic enzymes and cause SEVERE INFLAMMATION, including the hydrolysis of such structures such as collagen and cause a very painful situation -This is why you DON’T want to have high levels of uric acid -Gout is caused by excess levels of uric acid in the blood - It can result from several different defects: 1. It can be a defect in excretion of the uric acid 2. It can be an overproduction of purines that occurs -If one or the other or a combination of the two occurs, uric acid accumulates to extremely high levels -When uric acid is collected by the kidneys and it is taken up by the kidney, remember that urine tends to have a LOW pH so we’re going to see uric acid in the noncharged form as depicted. -In urine uric acid forms these microcrystalic structures. If the kidney has an especially acid pH the uric acid can be the basis of forming KIDNEY STONES |
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The salvaging enzymes – how do they work? Why are they medically relevant?
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-one example is this enzyme called adenosine phosphoribosyl transferase
-what it’s going to do is take preformed adenine (typically from the diet) and attach it to a ribose 5 phosphate to make AMP -Get a nucleophilic attack at the position indicated by the red arrow, liberating pyrophosphate, which then immediately hydrolyzes to two phosphates and we generate AMP **we didn’t have to do it de novo so we don’t have to expend a ton of ATP and use up a bunch of raw materials. This is a tremendous advantage to a somatic cell not to have to make this nucleotide de novo.** |
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How does the brain make purine nucleotides?
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-the liver is constantly in the mode of making lots of purine nucleotides and then degrading them to either hypoxanthine or guanine
-if it does not convert it to uric acid, then that hypoxanthine or guanine can be released into the blood stream -the extrahepatic tissues take advantage of the purines produced by the liver by using this salvaging enzyme called HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE (HGPRTase) -this is the most medically relevant enzyme!! -this is the same reaction we saw before: -->take base, take PRPP, promote nucleophilic attack at the 1 position, attach the base to the ribose-5-phosphate, and liberate pyrophosphate. **This is especially important in NEURAL cells because the brain doesn’t want to be expending a lot of ATP and using up a lot of raw materials to be making ATP. ATP levels have to be high for the brain to operate normally. This will become a very predominate mechanism by which brain cells will make purine nucleotides to support its metabolic activities** |
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What is the rationale for treatment of Cancer and Viral Infections
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-In cancer, we have cells that are replicating out of control
-In viral infections, if we have herpes infection for example, the virus is replicating its DNA at a very rapid rate -Most cells in the patient are in the resting, non-replicating state -->Strategy: interfere with nucleotide metabolism and the starting materials for DNA replication! -Thus, we are able to more specifically kill cancer/virus infected cells or at least to retard the replication in those cells -BUT there are some toxic effects with interfering with nucleotide metabolism -->The patient will need replicating cells for normal physiological function -->There are also some other unintended effects The problem: How to minimize detrimental effects of treatment? How do we NOT affect cells that we need to replicate for normal physiological function? |
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**Don't forget to look over the drug/disease flashcard sets!
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You've got this !!
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