Structural Studies of Human 5'-Nucleotidases

Abstract: 5’-Nucleotidases (5’NTs) are catabolic enzymes of the nucleotide metabolism. They catalyze dephosphorylation of deoxyribo- and ribonucleoside monophosphates and constitute an important control point in the regulation of intracellular nucleotide pools for the maintenance of correct DNA and RNA synthesis.By removing the alfa-phosphate group from a nucleotide, the 5’NTs release the nucleoside to pass the plasma membrane by facilitated diffusion. Depending on the cellular need for nucleotides, the nucleosides can either exit the cell for reuse elsewhere or be imported and subsequently phosphorylated by nucleoside and nucleotide kinases.The knowledge of how nucleotides are metabolized has been used for rational design of nucleoside analogues that are used in treatment of cancer and viral diseases. These drugs are phosphorylated within the cell to become active. Their dephosphorylation by 5’NTs might be one of the mechanisms behind the resistance experienced by patients towards such drugs.This thesis describes structure-function studies on four of the seven known human 5’-NTs. The focus of the work is on the substrate specificity and regulation of these enzymes. Inactive variants of the mitochondrial and cytosolic deoxynucleotidases and the cytosolic 5’-nucleotidase II were used to characterize the structural basis for their substrate specificity in high detail.Based on structures of the apoprotein and activator/activator+substrate complexes of cytosolic 5’-nucleotidase II, a mechanism for the allosteric activation of this enzyme was presented. In this mechanism, the activator induces a conformational change that involves conserved residues of the active site. The conformational change drastically increases the enzyme affinity for the phosphate moiety of the substrate.