Purines are primarily synthesized in the liver, specifically in the cytosol. The first “fully-formed” nucleotide is Inosine Monophosphate (IMP). IMP is formed from Phosphoribosyl Pyrophosphate (PRPP) and leads to AMP and GMP. Stop and stare into that wonderful chart above (from the OSU website).
This is IMP. It has a purine and a ribose.
PRPP is manufactured via the Pentose Phosphate Pathway. PRPP, the source of the sugar for purine nucleotide, is synthesized from ribose 5-phosphate.
The purine ring system of IMP is built from the PRPP molecule in an 11-step process. Five of these are analogous, starting with the activation by phosphorylation of a carbonyl oxygen. Essentially, the ring system is built by a series of small steps, one atom at a time. The process was discovered in 1948 by John Buchanan using isotope tracing.
In this lecture he walks through each of the 11 steps:
- PRPP serves as a scaffold for the synthesis. First N supplied by AA, Glu.
- Gly is activated by ATP: growing ring contain 2 N, 2 C
- N10-formyl THF contributes on formyl group
- Carbonyl group carried by the residual Gly is activated by ATP. The O2 is replaced by an amino group given by Glu
- The Carbonyl group carried by the formyl group is activated by ATP. It’s a dehydration reaction leading to the closure of the ring, the Imidazole
- A molecule of bicarbonate is activated by ATP. This adds one carboxylate group to the ring system
- This group is transferred to a nearby carbon on the imidazole ring
- The carbonyl group carried by the carboxylate is activated by ATP and a molecule of Asp is covalently bond to the activated carbon
- The carbon skeleton of Asp in Step 8 is removed leaving behind an amino group. Fumarate is released
- Final Carbon is contributed by N10-formyl THF
- A dehydration reaction leads to the closure of the second ring
A diagram from the OSU website:
Just know that the ingredients include Glutamate and Glycine (amino acids) along with bicarbonate (HCO3-) and Folate. Requires the hydrolysis of six ATP.
The enzyme catalyzing the IMP synthesis are organized into a multi-protein complex.
“This is another example of substrate channeling to increase biosynthesis efficiency and prevents diffusion and degradation of the unstable intermediates.” -Dr. Viel
IMP Conversion: AMP and GMP Synthesis
IMP is a branch point in purine biosynthesis. The conversion of IMP can lead to either GMP or AMP (you know, the precursor to ADP and ATP) via distinct pathways. These conversions involve replacing one substituent of the purine ring with another chemical group (which resembles Steps 8/9 of IMP synthesis).
For AMP, the carbonyl oxygen is replaced by an amino group. For GMP, an amino group replaces one H carried by the second ring.
AMP: First, the carbonyl O carried by IMP is “activated” by GTP and replaced with Asp. This is called adenylosuccinate. The second step cleaves off the fumarate portion of Asp leaving what is AMP.
GMP: IMP is oxidized to form XMP coupled with the reduction of NAD+. The carbonyl O is activated by ATP and an amino group is transferred from a Glutamine, releasing AMP and Glutamate.
The synthesis of AMP requires GTP while GMP requires ATP. This helps keep the two balanced. Namely,
“The accumulation of excess GTP will lead to accelerated AMP synthesis from IMP instead, at the expense of GMP synthesis. Conversely, since the conversion of IMP to GMP requires ATP, the accumulation of excess ATP leads to accelerated synthesis of GMP over that of AMP.” — Medical BioChemistry Page
Purine Biosynthesis Regulation
The three main points of regulation:
First is the transfer of an amido group to PRPP catalyzed by glutamine PRPP amidotransferase. Controlled by feedback inhibition: IMP and the end products AMP and GMP act as inhibitors. These also inhibit the PRPP synthase.
The second control point. Excess of AMP inhibits the synthesis of AMP on the IMP -> AMP branch pathway. Similarly, GMP in inhibits IMP -> GMP.
Third, is the regulation by GTP and ATP mentioned above.
UMP is assembled in its entirety and then attached to PRPP. Ingredients include Carbamoyl Phosphate, Asparate and NAD+. Carbamoyl Phosphate and Asp react to form N-cabamoyl aspartate controlled by Aspartate Transcarbamoylase (ATCase)–see Allosteric Enzyme lecture. This is inhibited by the pyrimidine CTP.
UMP is converted to UTP by phosphorylation. UTP is modified to form CTP. UTP was the star player in the glycogen synthesis, if you recall. CTP will be a star player in the lipid metabolism, coming soon.
ATCase allosteric regulation
Hyperbolic graph of Initial Velocity vs [Asp]. Inhibited for high CTP. If both ATP and CTP are high, the inhibitory effects of CTP are countered. ATP and CTP bind to the regulatory subunits of the ATCase.
When the purine [ATP] is high, the pyrimidine production increases to help maintain purine-pyrimidine balance.
Carbamoyl Phosphate + Asp -> Carbamoyl Aspartate -> Dihydroorotate -> Orotate (coupled with reduction of NAD+)
The enzymes for this sequence are all part of a protein complex called CAD. Another example of substrate channeling.
The Orotate ring is attached to Ribose via PRPP leading to UMP which is phosphorylated to form UTP. Gln and ATP interact to form CTP.
End products here are UTP and CTP; whereas during purine synthesis, the end products are AMP and GMP.
The synthesis of nucleotide monophosphate to nucleotide diphosphate is catalyzed by a class of enzymes called nucleoside monophosphate kinases. These are not used during glycolysis, et al. Those pathways start with the diphosphates, ADP and GDP.
transfer of phosphate from ATP to AMP to form ADP. In general, ATP is the phosphate donor for the conversion of mono to diphosphate.
ATP + AMP <—(adenylate kinase)—> 2 ADP
2 ADP <—(OP or glycolysis)—> 2 ATP
ATP + NMP <—(nucleotide monophosphate kinase)–> ADP + NDP
“In general, ATP is the phosphate donor for the conversion of nucleotide monophosphate to nucleotide diphosphate. The nucleoside monophosphate kinases are generally specific for a particular base but not specific for ribose or deoxyribose.” — Dr. V
Converting NDP to NTP
NTP(donor) + NDP(acceptor) <—(nucleotide diphosphate kinase)–> NDP(donor) + NTP(acceptor)
Nucleophile — attracted to a nucleus. Has a (full or partial) negative formal charge, or even a region of high electron density. Attracted to things that are positively charged.
Electrophile — attracted to an electrons. Has a (full or partial) positive formal charge, or even a region of low electron density. Attracted to things that are negatively charged.
Since electrons flow from a region of high electron density to a lower density region, negative charges attracted to positive charges (Coulomb’s Law), a nucleophile can attack a region of positive charge. This is called a nucleophilic attack.
C4(H3)2, a carbon bonded to three other carbons, which is a carbo-cation, functions as an electrophile on the central carbon. The oxygen on the ethanol functions as a nucleophile. So the oxygen “attacks” that central carbon.
Sidebar to the sidebar:
The Schwartz Rules of Organic Chemistry (five things you need to know)
- Valence electrons
- Acid-Base chemistry (follow protons)
- Redox Chemistry
Now it’s time to “review” Sn1 Reactions. in quotes because I’m learning biochem and ochem concurrently.
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