Editing of the GluR-B site of pre-mRNA by ADAR1 and ADAR2

Adenosine deaminases that act on RNA (ADARs) are a family of enzymes that convert adenosine to inosine within double stranded RNA. While it is known that this conversion occurs by hydrolytic deamination, the exact mechanism is not yet known. This thesis describes studies aimed at the long-term goal of co-crystallizing ADAR1 and ADAR2 with a biological relevant substrate such as the R/G hairpin duplex. A substrate that contains a transition state analog in place of the R/G site adenosine will be tightly bound to ADAR, and therefore should co-crystallize. A Co-crystal will lead to a better understanding of deamination mechanism of ADAR. The studies with ADAR1 focus on the use of abasic site within the substrate, whereby adenosine and potential transition state analogs could be "floated" into a hole in the helix created by the abasic site. Initial result show that ADAR1 may deamiinate free floating AMP at low levels. This low-level deamination was not encouraging enough to pursue at this time. Studies with ADAR2 focus on determinin Km and Vmax through steady state kinetics. These experiments indicate that ADAR2 is subject to sever substrate inhibition. Km was determined to be 1.02 nM; Vmax was determined to be 1.08 x 10[-7) nmol/sec, and Ki was determined to be 5.26 nM.