Purine and Pyrimidine Metabolism Two pathways De novo pathway purines and pyrimidines o Highly conserved in eukaryotes Salvage pathway o Synthesis of nucleotides from starting materials amino acids ribose CO2 etc to make o Recovery of bases for making new nucleotides from other components o More divergent varied De Novo pathways purine and pyrimidine Purine pathway o Know the important components that go into making the nucleotide o 6 components PRPP glutamine aspartate Co2 glycine formate produces inosine monophosphate IMP PRPP phosphopyrosylphosphate o Purine cube cube of pathway reactions for de novo and salvage degradation IMP inosinate is the precursor for AMP and GMP o From the de novo pathway you take the 6 building blocks and start making nucleotides Adenylate AMP o Adenosine no phosphate on ribose Nomenclature Base is the aromatic part side means no phosphate o Nucleoside is without a phosphate tide means phosphosugar o Nucleotide contains a phosphate group Origin of Atoms Purine o Numbering begins on N N1 o Goes counterclockwise o N are always odd N1 3 7 9 o Link to ribose is N9 o N1 comes from Aspartate o C2 from formate o N3 Glutamine amide o C4 C5 Glycine o C6 HCO3 o N7 same glycine as C4 C5 o C8 from Formate o N9 from glutamine amide Pyrimidine o Numbering begins on N N1 o Goes clockwise o N are always odd N1 3 o Link to ribose is N1 o N1 from Aspartate o C2 from HCO3 o N3 from glutamine amide o C4 C5 C6 from same aspartate as N Bases are numbered without prime o Sugar ring is numbered again starting at 1 with Starts at the site of attachment to the purine pyrimidine Attachment occurs by a beta glycosidic linkage o Only when numbering nucleoside or nucleotide All sugars are D in the furanose conformation o Amino acids are L Once the base is added the nucleotide is held in the beta glycosidic form Numbering hints For pyrimidines nitrogens are always at odd positions 1 3 not 5 o Attached to the sugar at N1 For purines nitrogens are always at odd positions 1 3 7 9 o Attached to the sugar at N9 Know the naming of the nucleotides based on base nucleoside and nucleotide Naming of bases Base Adenine Guanine Cytosine Uracil Thymine Nucleoside Adenosine Guanosine Cytidine Uridine Deoxythymidine Nucleotide Adenylate Guanylate Cytidylate Uridylate Deoxythymidylate Other Metabolites similar with purine pyrimidine rings A G C found in DNA and RNA U only in RNA T only in DNA Base nucleoside nucleotide Hypoxanthine inosine inosinate IMP Xanthine Xanthosine Xanthylate XMP Orotate Orotidylate OMP Uric Acid Nucleotide derivatives o Caffeine Tri methyl Xanthine o Theobromine in chocolate Di Methyl Xanthine o Anti HIV ddC and AZT o cAMP SAM NAD NADP UDP glucose CoA FAD Biosynthesis of purine Assembled on a scaffold of ribose phosphate o Build at N9 since it is the site of attachment to the ribose Uses 7 high energy phosphate bonds over 11 steps to make inosine monophosphate IMP Regulation at steps 1 and 2 o Pathway becomes committed at step 2 once you ve made the phosphoribosylamine Loss of pyrophosphate is irreversible since pyrophosphate is not generally found in the cell so it won t go back on 1 5C phosphate PRPP o 5C ring with phosphate on C5 o Conformation around anomeric carbon is alpha Since there will be inversion in a following step o Use ATP to AMP to add pyrophosphate making PRPP 5 phosphoribosyl alpha pyrophosphate PRPP 2 PRPP 5 phosphoribosylamine o Pyrophosphate loss glutamine glutamate using amidophosphoribosyl transferase Makes inversion and beta phosphoribosylamine Irreversible because of loss of pyrophosphate Glutamine acts as a donor of N amine group Nitrogens can only come from the side chain of glutamine or the backbone of aspartate o Glutamine glutamate o Aspartate fumarate o Addition uses a nucleotide and makes a phosphoester intermediate High energy phosphate for SN2 addition of NH2 3 5 phosphoribosylamine Glycinamide ribotide GAR o Requires ATP o Addition of glycine to alpha amine 4 GAR formyl GAR FGAR o Addition of formyl group from Tetrahydroformate THF Basically aldehyde gets added to NH2 on end of glycine 5 FGAR FGAM o Substitution of NH2 for the ketone on first carbon of glycine o Requires glutamine and ATP 6 FGAM 5 aminoimidazole ribotide AIR o Uses an enamine reaction to accomplish ring closure o Uses ATP 7 AIR carboxy AIR CAIR o Use of ATP to add HCO3 COO group to C4 on the ring 8 CAIR SACAIR 5 aminoimidazole 4 N succinylcarboxamide ribotide o Addition of Aspartate using ATP Intermediate to add an amine to the carboxy added from HCO3 in step 7 9 SACAIR AICAR 5 aminoimidazole carboxamine ribotide o Loss of fumarate no energy required 10 AICAR FAICAR o Addition of formyl group to amine N1 making C2 11 FAICAR IMP o Loss of water ATP required at steps 1 3 5 6 7 8 THF Tetra hydrofolate Enzyme carrier of carbon o Versatile covers them in different oxidation states and chemical forms o 3 oxidation steps o 5 different chemical form Comes from folic acid in leaves vitamin B9 Has methylpterin and aminobenzoic acid rings that contain the carbons o Has glutamates to allow enzymes to hold onto THF o Carbon can be attached on the N5 and the N10 position Oxidation states of carbon Most reduced most oxidized Methanol Formaldehyde Formate Methanol o Carries methyl group o THF N5 methyl THF Formaldehyde o Carries methylene group o THF N5 N10 methylene THF Formate o Carries formyl aldehyde formimino CH NH methenyl CH C o N5 formyl N10formyl o N5 formimino o N5 N10 methenyl N10 formyl is used in purine synthesis de novo pathway o For steps 4 and 10 IMP AMP GMP AMP GMP Regulation Purine base intermediate Branch point for AMP and GMP synthesis o Change the C6 ketone to an amine o Done through the use of aspartate and GTP o Loss of fumarate yields AMP o Addition of amine onto C2 o Use of NAD NADH oxidizing power Produces XMP which then uses glutamine and ATP to make GMP Use of opposite xMP molecule helps balance the cellular levels of AMP and GMP Nucleotides are going to inhibit the synthesis of PRPP and 5 phosphoribosylamine PRPP activates purine synthesis feedforward AMP and GMP feedback inhibit on their own pathways probably to balance the levels in the cell for both ADP and GDP inhibit PRPP synthesis Lecture 2 January 14 Pyrimidine Biosynthesis AICAR is an analog of AMP and has physiological effects which results in muscle building The deNovo pathway for pyrimidine synthesis makes oritidic acid which then makes UMP Pyrimidine The N1 and C4 6 comes from aspartate C2