Enzymatic Characterization Of The Ammonia Tunnel In Helicobacter Pylori Asp-Trnaasn/glu-Trnagln Amidotransferase

<p>The <i>Helicobacter pylori</i> (<i>H. pylori</i>) Asp-tRNA^Asn/Glu-tRNA^Gln amidotransferase (AdT) plays important roles in indirect aminoacylation and translational fidelity; however, its inter-domain communication and ammonia delivery mechanisms are not well understood. In the present study, we investigated the three activities of <i>H. pylori</i> AdT (glutaminase, kinase and transamidase) and used these reactions as probes to examine the inter-domain communication and ammonia delivery mechanisms between this enzyme's two isolated active sites. We adapted and optimized an assay to kinetically characterize a series of mutations at conserved positions throughout the putative AdT ammonia tunnel. The kinase assay enabled us to identify mutations within AdT, specifically T149 and K89, for further enzymatic characterization and molecular dynamics (MD) simulations and correlation analyses to unveil a set of 59 residues that may form the interdomain communication pathway between AdT's two active sites. The glutaminase and transamidase assays identified another residue, D185, in the GatA subunit. Kinetic and computational characterizations of D185 AdT mutants suggest that D185 serves as a general acid or base in ammonia delivery. These results are the first demonstration of acid/base chemistry within an ammonia tunnel. Finally, preliminary characterization of the predicted ammonia tunnel gate residues (K89 and E126 in the GatB subunit) suggest that proper positioning of the appropriate charge states in the tunnel are important for AdT catalysis. The results presented in this dissertation extend our understanding of AdT's distinct ammonia transfer mechanism.</p>

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Open Access Dissertation

Discipline thesis:degree_discipline

Chemistry

Year dc:date.available

2013

Author dc:creator

• Zhao, Liangjun

Contributors dc:contributor

• Tamara L. Hendrickson

• AMIDOTRANSFERASE
• AMMONIA TUNNEL
• HELICOBACTER PYLORI
• Biochemistry

Repository record dc:identifier

https://digitalcommons.wayne.edu/oa_dissertations/867

OAI identifier oai:identifier

oai:digitalcommons.wayne.edu:oa_dissertations-1866