Nucleotide Metabolism Nucleotide sources In humans dietary nucleotide bases are rarely incorporated into nucleotides As a result humans must synthesize their own nucleotide bases With the exception of a few parasitic prokaryotes all organisms can synthesize nucleotides Although all nucleated eukaryotic cells can synthesize nucleotides most human synthesis occurs in the liver Nucleotide synthesis is tightly regulated Nucleotide synthesis is somewhat expensive in that the pathways use several molecules with other uses In addition although pyrimidines can be degraded into standard metabolic intermediates purine catabolism does not alter the basic purine structure and excessive levels of purines can be toxic Purine synthesis Purine production occurs primarily in the liver although most tissues produce at least small amounts Purine biosynthesis begins with ribose 5 phosphate a product of the hexose monophosphate pathway and ends with the hypoxanthinecontaining inosine monophosphate Both of the first two reactions in the purine biosynthetic pathway are tightly regulated The first reaction of the purine synthesis pathway catalyzed by ribose 5phosphate pyrophosphokinase produces phosphoribosylpyrophosphate PRPP PRPP is not committed to purine biosynthesis it is also used for other processes such as pyrimidine biosynthesis histidine biosynthesis and nucleotide base salvage However availability of PRPP is critical for purine biosynthesis The second step the loss of the PRPP pyrophosphate and the addition of an amino group from glutamine is catalyzed by PRPP glutamyl amidotransferase This step is the committed step for purine biosynthesis and is also a regulated step The pathway includes nine additional enzymatic reactions it uses the ribose 5phosphate as a platform for the construction of the purine ring structure The pathway first builds the 5 membered ring and then builds the six membered ring As is generally the case in biosynthetic reactions free ammonium is not used as a nitrogen donor in the pathway instead the requisite organic nitrogen is obtained from the amino acids glutamine glycine and aspartate One of the reactions in the pathway involves the incorporation of carbon dioxide the carboxylase responsible for the reaction is somewhat unusual in that it is not a biotin dependent enzyme Copyright 2000 2003 Mark Brandt Ph D 54 The product of the purine biosynthesis pathway is inosine monophosphate IMP which contains the base hypoxanthine Copyright 2000 2003 Mark Brandt Ph D 55 Three of the ring atoms in purines are derived from glycine the remaining six ring atoms come from six different molecules The purine structure includes nitrogens derived from glutamine yielding glutamate and aspartate yielding fumarate One carbon in the purine ring comes from carbon dioxide Two of the carbons in the purine structure come from N10 formyl tetrahydrofolate which is one reason why tetrahydrofolate is so important for nucleotide biosynthesis Ribose 5 phosphate pyrophosphokinase is feedback inhibited by nucleoside diphosphates especially ADP and GDP and dTDP PRPP Glutamyl amidotransferase is inhibited by any adenosine or guanosine phosphate It is stimulated by its substrate PRPP which is an important factor in regulation of purine synthesis Regulation of purine metabolism will be discussed further below AMP and GMP synthesis Inosine monophosphate does not accumulate instead it is rapidly converted to adenosine monophosphate AMP and guanosine monophosphate GMP Each of these processes is comprised of two reactions The first step in each pathway is inhibited by the final product IMP dehydrogenase by GMP and adenylosuccinate synthetase by AMP Note that ATP is required for GMP synthesis and GTP is required for AMP synthesis in addition each nucleoside triphosphate acts as a stimulator of the synthesis of the other purine derivative allowing high levels of one purine nucleotide to favor diversion of IMP toward production of the other Copyright 2000 2003 Mark Brandt Ph D 56 The second enzyme in the AMP pathway adenylosuccinate lyase catalyzes a similar reaction in the IMP pathway In both cases the reaction involves the cleavage of the aspartate added releasing fumarate while leaving the aspartate nitrogen attached to the base The base in xanthosine monophosphate is xanthine Fumarate is generated from aspartate as part of the IMP synthesis pathway and during conversion of IMP to AMP This pathway can result in physiologically significant levels of fumarate release in addition muscle may cycle AMP back to IMP to enhance fumarate generation Fumarate generation increases the net amount of carbon in the TCA cycle NTP generation The purine nucleoside monophosphates produced in the biosynthetic pathway need to be converted to nucleoside triphosphates Enzymes specific for the nucleoside monophosphate catalyze the conversion of the monophosphate to the diphosphate Adenylate kinase is specific for AMP and guanylate kinase is specific for GMP The enzyme nucleoside diphosphate kinase allows donation of terminal phosphate from any NTP to any NDP Because free ATP is present in higher levels than any other nucleotide and because ATP can be generated by metabolic processes by the substrate level phosphorylation steps of glycolysis and by oxidative phosphorylation ATP is usually used as the phosphate donor GTP is occasionally used also because it can also be generated from GDP by the succinylCoA synthetase reaction of the TCA cycle Salvage pathways Although humans tend not to use dietary nucleotides for nucleic acid synthesis humans do recycle nucleotides and free nucleotide bases in circulation This important process is termed the purine salvage pathway It is important because free purines are somewhat toxic and because the liver which synthesizes most nucleotides releases the compounds as either free bases or free nucleosides Copyright 2000 2003 Mark Brandt Ph D 57 Free purines are attached to the ribose ring using PRPP as the base acceptor by two enzymes Adenine is attached by adenine phosphoribosyl transferase Both hypoxanthine and guanine are attached to the ribose ring by hypoxanthineguanine phosphoribosyl transferase HGPRT The salvage pathway decreases the levels of PRPP and therefore decreases the rate of purine synthesis This is an important regulatory mechanism for purine metabolism A second pathway allows recycling of nucleosides by initiating the process of adding the 5 phosphates Adenosine kinase is specific