Purine and Pyrimidine Synthesis
What is the role of 'CPSII' in UMP synthesis in eukaryotic cells?
"CPSII" is the CPS catalytic site on CAD in the inner mitochondrial membrane in eukaryotic cells. It is an enzyme that catalyzes the reactions that produce carbamoyl phosphate in the cytosol from glutamine and bicarbonate. In pyrimidine biosynthesis, it serves as the rate-limiting enzyme and is highly regulated. It is activated by ATP and PRPP and it is inhibited by UMP (the end product of the pyrimidine synthesis pathway).
What are the amino acid sources for the NH2 come from to form AMP & GMP from IMP?
AMP synthesis uses aspartate as it is converted to fumarate via the Urea cycle and GMP synthesis uses glutamine as it is deaminated to form glutamate.
How do the concentrations of ATP, CTP and UTP affect the activity of ATCase?
ATCase controls the rate of pyrimidine biosynthesis by altering its catalytic velocity in response to cellular levels of both pyrimidines and purines. The end-product of the pyrimidine pathway, CTP, induces a decrease in catalytic velocity, whereas ATP, the end-product of the parallel purine pathway, exerts the opposite effect, stimulating the catalytic activity. If both ATP and CTP are present, the enzyme is stimulated by ATP and is active. UTP can bind to the allosteric site, but inhibition of ATCase by UTP is possible only in combination with CTP.
Describe the unusual proton transfer from dihydroorotate dehydrogenase to CoQ that occurs in eukaryotes.
DHODH is a flavoprotein, meaning it contains an FMN prosthetic group. Dihydroorotate dh oxidizes dihydroorotate. The enzyme contains FMN, FAD, and a 2Fe-2S cluster. The protons is transferred to FMN to form FMNH2. The FMNH2 is oxidized by reduction of FADH2, whose electrons are transferred to Q cycle, allowing the proton to be passed to CoQ.
What is the purine cycle in muscle cells? What is it relationship to the TCA cycle?
Due to dearth in mitochondria in the muscle cells (as opposed to the liver) de novo synthesis is exceptionally it occurs in the myocyte's cytosolic compartment. The phosphorylation and dephosphorylation of 2 ADP's to produce 1 ATP and 1 AMP is catalyzed by adenylyl kinase/myokinase. Because fumarate is generated from aspartate (aspartate → argininosuccinate → fumarate) it can be used as an intermediate in the TCA cycle, as well as increase the rate of oxidative phosphorylation in skeletal muscle.
Describe the role of HGPRT and APRT in recycling purines.
HGPRT is a transferase that catalyzes conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate. This reaction transfers the 5-phosphoribosyl group from 5-phosphoribosyl 1-pyrophosphate (PRPP) to the purine. HGPRT functions primarily to salvage purines from degraded DNA to reintroduce into purine synthetic pathways. APRT catalyzes Adenine + PRPP → Adenylate (AMP) + PPi. Although APRT is functionally redundant in these organisms, it becomes more important during periods of rapid growth, such as embryogenesis and tumor growth.
How is HGPRT the major deletion mutation that is central to forming a hybridoma that produces a monoclonal antibody?
HGPRT is an important enzyme in the salvage pathway of purines. The deletion in the myeloma cells for the HGPRT gene so that they cannot use the salvage pathways and they are also mutated such that they cannot do de novo either. The myeloma cells are an immortal cell line. Then, the myelomas are fused with spleen cells. The spleen cells are making the monoclonal antibody. The media has hypoxanthine, which can only be used by cells with HGPRT. The fused cell line has the immortal cell line from the myelomas and the HGPRT from the spleen cells. In the media is the drug aminopterin, which is an inhibitor of DHFR, so you cannot replicate. The main idea is with hypoxanthine in the medium, only cells with HGPRT will survive. SO only the fused hybridoma survive because they are immortal and have HGPRT.
How is pyrimidine biosynthesis regulated in bacteria and eukaryotic animal cells?
In bacteria pyrimidine biosynthesis is regulated by ATCase Rxn 2, control is exerted by the allosteric stimulation of ATP and it is inhibited by CTP. In many bacteria though UTP is an inhibitor. In animal cells ATCase is not regulatory, rather pyrimidine biosynthesis is controlled by the activity of CPSII. Another level of control is in the formation of OMP, and due to the availability of PRPP. Pyrophosphate kinase is inhibited by ADP and GDP.
Describe ATCase structure from prokaryotes.
In prokaryotes, ATCase is an independent enzyme that is not part of a complex, unlike the ATCase domain in CAD in eukaryotes. ATCase is inhibited by CTP and stimulated by ATP. The ATCase in prokaryotes has allosteric regulatory sites and active sites. The allosteric site, found in the allosteric domain of the R chains of the ATCase complex binds to the nucleotides ATP, CTP and/or UTP. There is one site with high affinity for ATP and CTP and one with 10- to 20-fold lower affinity for these nucleotides in each regulatory dimer. The enzyme also has a zinc domain in its R chain. One domain of ATCase is made up of one catalytic chain (C1) and its associated regularly is composed of two independently folding domains: the aspartate-binding (Asp) domain and carbamoyl phosphate-binding regulatory chain is composed of the zinc-binding (Zn) domain and nucleotide-binding (allosteric) domain. The enzyme exists in a R conformation that is active or in an inactive T conformation, which is induced by binding of CTP.
Describe the evolutionary changes that occur to the proteins pyrimidine pathway structurally from prokaryotes to eukaryotes?
In prokaryotes, the enzymes that contribute to pyrimidine synthesis are separate in the cytosol, but in eukaryotes the first three enzymes in pyrimidine synthesis, CPS, ATCase and dihydroorotase, form a complex called CAD. CAD is found on the cytosolic side of the inner mitochondrial membrane. In prokaryotes, ATCase is the controlling enzyme of pyrimidine synthesis, while in eukaryotes the CPS domain of CAD is the controlling enzyme.
How does UMP synthesis differ in prokaryotes and eukaryotes?
In prokaryotes, the first several enzymes lay around in cytoplasm. In eukaryotes, the first 3 enzymes (carbamoyl phosphate synthetase, aspartate carbamoyltransferase, and dihydroorotase) are part of a single trifunctional protein complex known as CAD. Thus, the bacterial ATCase catalyzes the first committed step in the pyrimidine pathway and is allosterically regulated. In contrast, there are two CPSases in mammalian cells, CPSII, the CAD activity committed to pyrimidine biosynthesis, and CPSI, an ammonia-dependent enzyme that initiates urea biosynthesis in the mitochondria. Because CAD CPSase catalyzes the first step in the pathway, ATCase is unregulated and there is no counterpart of the regulatory chain found in E. coli ATCase. Whereas the active site of E. coli DHOase has two zinc ions and a carboxylysine that bridges the metal centers, the mammalian DHOase domain probably belongs to a different subgroup of the amidohydrolase superfamily.
How does the ribosylation differ in purines and pyrimidine synthesis?
In pyrimidine synthesis, the ring structure is made first, and then attached to a ribose sugar base (PRPP). In purine synthesis, the purine ring structure is build directly onto the ribose base (PRPP).
Discuss the regeneration of N5N10MTHF from the serine-glycine complex and the methylation of dUMP to form dTMP.
In the methylation of dUMP to dTMP catalyzed by thymidylate synthase, N5N10MTHF is the methyl group donor. By losing it's methyl group, N5N10MTHF is converted into dihydrofolate. Dihydrofolate can be recycled and used to synthesize tetrahydrofolate. The reduction of DHF to THF is catalyzed by dihydrofolate reductase and used an NADPH.Tetrahydrofolate is then used as a methyl acceptor in the conversion of serine to glycine, catalyzed by serine hydroxymethyltransferase.
Explain the roles of NDPK and CrK in cells. Where are these enzymes located in cells? In the case of CrK one of it locations is very unique, explain the phosphocreatine cycle.
NDPK is found in the mitochondria and in the soluble cytoplasm. NDPK is the source of RNA and DNA precursors, except ATP. NDPK are involved in the synthesis of nucleoside triphosphates (NTP), such as guanosine triphosphate (GTP), cytidine triphosphate (CTP) and uridine triphosphate (UTP). NDPK's roles in these NTPs differ; generally, kinases bring in NTPs for nucleic acid synthesis. CTP is provided for lipid synthesis, UTP for polysaccharide synthesis while GTP is used for protein elongation and signal transduction. During cAMP-mediated signal transduction, NDPK is responsible for phosphorylating GDP released from G proteins activated from receptor binding; once ATP donates a phosphate group via activity of NDPK, GTP is consecutively bound. CrK exists in the mitochondrion (inner membrane space) and in the microfibrils in the cytosol. Mitochondrial CrK functions to utilize ATP shuttled out of the matrix to the inner membrane space to phosphorylate creatine, making phosphocreatine, which is then shuttled out of the mitochondrion into the cytosol. There, phosphocreatine functions as storage of excess ATP (as storage of the phosphoryl group). When the cell is in need of ATP, microfibryl CrK dephosphorylates the phosphocreatine and phosphorylates ATP in the cytosol.
Explain the role of the following enzymes; nucleotide diphosphate kinase (NDPK) and creatine kinase (CrK)
Nucleotide diphosphate kinase catalyzes the exchange of a phosphate group between dinucleotides and trinucleotides reversibly. It is nonspecific. Creatine kinase catalyzes the phosphorylation of creatine to phosphocreatine using ATP. Phosphocreatine serves as a source of a phosphoryl group for the quick regeneration of ATP in the cytosol.
How is the purine synthetic pathway controlled and regulated? This includes explaining feedback control in purine synthesis.
Purine synthesis is controlled by feedback control. First, PRPP synthesis is only active when ATP is coordinated with Mg2+. PRPP synthetase is regulated by phosphorylation and allosteric regulation. It is activated by presence of phosphate and inhibited by presence of ADP; it is suggested that phosphate and ADP compete for the same regulatory site. At normal concentrations, phosphate activates the enzyme by binding to its allosteric regulatory site. However, at high concentrations, phosphate is shown to have an inhibitory effect by competing with the substrate ribose 5-phosphate for binding at the active site. PRPP synthetase is also inhibited by AMP, IMP and GMP. The glutamine PRPP aminotransferase reaction is the committed step to purine synthesis and is also regulated by feedback. The enzyme is inhibited by IMP, GMP and AMP, the products of the pathway. IMP is the branch point for purine synthesis and can be formed into either AMP or GMP. AMP synthesis is inhibited by inhibition of adenylosuccinate synthase by feedback of AMP. GMP synthesis is inhibited by inhibition of IMP dehydrogenase by feedback of GMP.
In the synthesis of IMP, why is the second reaction the first committed step? What other pathways utilize PRPP?
The glutamine PRPP aminotransferase reaction is the committed step to de novo purine synthesis. The synthesis of PRPP via ribose phosphate pyrophosphokinase is not the committed step to purine synthesis because PRPP is utilized in pyrimidine synthesis as well. 5-phosphoribosyl-1-amine, the product of the committed step, is only used in the purine de novo synthesis pathway and, thus, is the committed step to IMP synthesis.
Describe the different mechanisms of amination of IMP to form GMP or AMP
The mechanism for the formation of AMP from IMP is as follows: first, IMP is condensed with aspartate via adenylosuccinate synthase, forming adenylosuccinate. Next, fumarate is cleaved from adenylosuccinate via adenylosuccinate lyase, which leaves adenylate phosphorylated, forming AMP. In order to transfer its amino group to IMP, aspartate must be condensed and cleaved. The mechanism for the amination of IMP to GMP is as follows: IMP is first oxidized to XMP via IMP dehydrogenase, producing an NADH and xanthosine monophosphate. XMP is aminated to GMP via Glutamate synthase transferring the side-chain amino group of glutamine to XMP.
What are the rate-limiting steps of purine synthesis?
There are is one limiting step for the synthesis of IMP. The committed step to purine synthesis is not rate limiting, (this is the glutamine PRPP aminotransferase reaction). The rate-limiting step in IMP synthesis is the cleavage of fumarate via adenylosuccinate synthetase and adenylosuccinate lyase to yield ribose 5-phosphate
Explain the T-> R transition in ATCase.
With CTP present, UTP binding is enhanced and preferentially directed to the low-affinity sites. On the converse, UTP binding leads to enhanced affinity for CTP at the high-affinity sites and together they inhibit enzyme activity by up to 95%, while CTP binding alone inhibits activity to 50% to 70%. Comparison of the crystal structures of the T and R forms of ATCase show that it swells in size during the allosteric transition, and that the catalytic subunits condense during this process. The two catalytic trimers move apart along the threefold axis by 12 Å, and they rotate about this axis by 5° each, ultimately leading to a reorientation of the regulatory subunits around their twofold axis by 15°. (after binding of UTP/CTP, the two catalytic trimers move apart and rotate slightly, leading to the inactive T state)