Question

For every pyrimidine mononucleotide synthesized de novo, 5 ATPs are consumed. For every purine nucleotide synthesized de novo, 9 ATPs are consumed. Because of this high cost, it is essential to tightly control nucleotide synthesis to closely match the needs of the cell at any given time. When the energy charge of a cell is low, cells will not replicate DNA. There is no need for deoxyribonucleotides (dNTPs) so their synthesis is turned off. Conversely, when the energy charge is high, cells are primed to grow and divide. The dNTP synthesis is high to meet the demands of DNA synthesis. Ribonucleotide (NTP) synthesis also increases to satisfy RNA synthesis, glycogen synthesis (UTP), phospholipid synthesis (CTP) and the energy needs (ATP, and to a lesser extent GTP) of other biosynthetic pathways. Intricate feedback mechanisms ensure balanced pools of NTPs and dNTPs.

1.) Record the effect, on nucleotide end products, of elevated CTP and ATP on aspartate transcarbamoylase-mediated nucleotide biosynthesis.

allosteric effector	[ATP]	[GTP]	[CTP]	[UTP]
CTP
ATP

2.) Aspartate transcarbamoylase catalyzes the first committed step of de novo pyrimidine nucleotide synthesis. Propose a physiological rationale for its differential regulation by a purine nucleotide (ATP) and a pyrimidine nucleotide (CTP).

Answer 1

(1)ATcase can sebe literally separated into regulatory and catalytic subunits by treatment with a mercurial compound such as p-hydroxymercuribenzoate, which reacts with sulfhydryl groups . The sedimentation coefficient of the native enzyme is 11.6S, whereas those of the dissociated subunits are 2.8S and 5.8S, indicating subunits of different size. The subunits can be readily separated by ion-exchange chromatography because they differ markedly in charge or by centrifugation in a sucrose density gradient because they differ in size . Furthermore, the attached p-mercuribenzoate groups can be removed from the separated subunits by adding an excess of mercaptoethanol. The isolated subunits provide materials that can be used to investigate and characterize the individual subunits and their interactions with one another.

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(2).

A rate-limiting enzyme in the de novo synthesis of pyrimidines, is regulated through reversible phosphorylation, Myc-dependent transcriptional changes and caspase-mediated degradation. These studies point to increasing evidence for cooperation between key cell signaling pathways and basic elements of cellular metabolism, and suggest that these events have the potential to determine distinct cellular fates, including growth, differentiation and death. This review highlights some of the recent advances in the regulation of pyrimidine synthesis by growth-factor-stimulated signaling pathways.