I 26 1 Can Cells Synthesize Nucleotides There are two main ways to make purine and pyrimidine nucleotides 1 de novo meaning from scratch using ribose 5 phosphate as the starting material 2 Salvage pathways that can recover purine and pyrimidines that are obtained through the diet or degradation processes In terms of energy only the ribose can generate energy while the nucleotides cannot An example of this and how it is taken advantage of today is based on the difference between rapidly dividing cells and those that are slower In rapidly dividing cells a large amount of RNA and DNA is synthesized meaning that nucleotides need to be synthesized just as rapidly It is for this reason that these processes that create the nucleotides and the proliferating cells are constantly under study by researchers dealing with cancer II How Do Cells Synthesize Purines a History John Buchanan in 1948 was studying how nucleotides are synthesized by the body when he came upon the idea that birds excrete excess nitrogen in the form of uric acid which is a purine This implies a mechanism in the bird was used to create this nucleotide He then studied how this process worked by adding different components at different times to the bird to see how the atoms were discarded Uric acid is now used to understand how the atoms are distributed in the process Source of carbon and nitrogen atoms in the purine ring Uric Acid 5 C2 C8 N10 formyl THF b Order in which the groups are added 1 N1 aspartic acid 2 C4 C5 N7 Glcine 3 N3 N9 Glutamine 4 C6 CO2 2 C4 C5 N7 1 N9 3 C8 4 N3 5 C6 6 N1 7 C2 III IMP Synthesis Steps 1 11 1 Step 1 In order for the reaction to be initiated the ribose 5 phosphate must be activated This is done via the transfer of a pyrophosphoryl PP group from ATP to the C 1 carbon of the ribose 5 phosphate This is accomplished through PRPP synthetase and creates PRPP as the enzyme suggests INHIBITORS A PRPP is absolutely necessary in order to activate the beginning of purine synthesis It determines how quickly and often purines are synthesized Negative effectors actually act as feedback inhibitors that when in excess GDP and ADP the enzyme does not function Therefore this is the LIMITING STEP 2 Step 2 THIS IS THE COMMITED STEP As noted above the first group to be added to the ring is the N9 in uric acid This N9 is derived from the amino acid Glutamine which donates its NH2 group while accepting an OH group from H2O Therefore glutamine is reduced to Glutamate and Pyrophosphate This is catalyzed by Gln PRPP amidotransferase and forms Phosphoribosyl amine INHIBITORS A Gln PRPP amidotransferase is also subject to feedback inhibition but by all forms of the guanines GDP GTP GMP and all forms of the adenines ATP AMP ADP This occurs via specific allosteric sites on the enzyme that are specific for both guanine and adenine meaning when enough of both adenine and guanine nucleotides are made these products will bind to their individual site inhibiting the step B Another inhibitor of the enzyme is azaserine which is a glutamine ANALOG This is a molecule similar in structure to glutamine so that the enzyme often mistakes it for glutamine but when it binds the analog the enzyme becomes inhibited It does so more specifically by interacting with the nucleophilic groups surrounding the glutamine binding site on the enzyme C Folate analogs are used to inhibit purine formation and hence nucleic acid synthesis cell growth and cell divison Faster growing and dividing cells such as microbial cells are more susceptible to these analogs then normal cells and can therefore be used to inhibit the division of infectious bacteria and cancer They work by binding to DHFR which is necessary for the reduction of folate and hence the formation of THF needed in each mechanism 3 Step 3 The next molecule to be added is the C4 C5 N7 group These are derived from glycine and are added to the amine group N9 on the Phosphoribosyl amine by the ATP driven condensation of glycines carboxyl group with the amine This reaction is catalyzed by GAR synthetase and creates GAR This reaction proceeds in two steps 1 ATP phosphorylation of the carboxyl group on glycine activates it 2 amide bond is formed between the carboxyl and amine 4 Step 4 The fifth molecule to be added is C8 which is derived from N10 formyl THF In this step the N10 formly portion of the molecule is transferred via GAR transformylase to the amine group N7 This ends up forming an open ended ring but places the molecules in the right spot for the closing of the ring This creates FGAR the F standing for the formyl that was transferred 5 Step 5 The sixth molecule to be added is N3 and is obtained from glutamine like N9 was Hence the enzyme to catalyze the transfer of the amine group to the C5 carbon is yet another amidotransferase known as FGAR amidotransferase This then yields FGAM The reaction requires the use of both ATP and H20 6 Step 6 It is this step that leads to the formation of the imidazole ring It requires ATP to phosphorylate the oxygen atom of the formyl group and AIR synthetase is used to create AIR which visually shows the transfer of the double bond from between C5 and N3 to C4 and C5 while the ring is closed with a double bond formation between N7 and C8 7 Step 7 The seventh molecule to be added is C6 which is derived from CO2 CO2 is added to C4 which will form the C6 of the ring structure It is catalyzed by AIR carboxylase and uses ATP to create CAIR the C stands for the CO2 added to AIR 8 Step 8 The eighth molecule to be added is N1 derived from aspartate It is added to the C6 obtained in step 7 via the use of SAICAR Synthetase and ATP hydrolysis which catalyzes the condensation of Aspartate with CAIR This produces SAICAR 9 Step 9 The four carbons from aspartate that were bound with the amino group are removed as fumurate via adenylsuccinate lysase This leads to the formation of AICAR 10 Step 10 The ninth and final molecule added is C2 which is derived from N10 formyl THF AICAR transformlase catalyzes this transformation to be added to N1 to form FAICAR 11 Step 11 The final step in the reaction involves the dehydration of the formyl group added to create the bond between N1 and C2 closing the ring and releasing H20 This is accomplished through IMP synthase to produce IMP which serves as the precursor for both AMP and GMP synthesis OVERALL 6 ATPs are required Steps 1 3 5 6 7 and 8 Regulation occurs in the first two steps stating above the multiple inhibitors and the