Date of Award

Summer 8-2008

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry and Biochemistry

Committee Chair

Dr. Robert Bateman

Committee Chair Department

Chemistry and Biochemistry

Committee Member 2

Dr. Sabine Heinhorst

Committee Member 2 Department

Chemistry and Biochemistry

Committee Member 3

Dr. Wei Guo

Committee Member 3 Department

Chemistry and Biochemistry

Abstract

Sequence analyses revealed two putative glutaminyl cyclase genes in Drosophila melanogaster with greater than 60% amino acid sequence identity to previously identified glutaminyl cyclase in humans. Expression, purification, and characterization of both putative proteins revealed two enzymatically active glutaminyl cyclase enzymes. Comparison of the Drosophila melanogaster glutaminyl cyclase enzymes to the published human glutaminyl cyclase revealed the enzymes were very similar. The Km values obtained were identical to those of human glutaminyl cyclase for three different substrates tested. However, the Drosophila glutaminyl cyclases demonstrated lower turnover numbers. Both Drosophila glutaminyl cyclase isoforms were inhibited by imidazole, imidazole derivatives, and metal chelators. The predicted tertiary structure of the Drosophila glutaminyl cyclase isoforms revealed a conservation of the overall tertiary structure and active site amino acids when compared to human glutaminyl cyclase. Localization studies demonstrated differential location of the two isoforms within the Drosophila melanogaster embryo. One isoform was expressed at much higher levels and found within the developing nerve cord suggesting this isoform of the Drosophila glutaminyl cyclase is important in neuronal development. These glutaminyl cyclase isoforms join peptidyl-glycine a-amidating monooxygenase and prohormone convertase 2 as the known bioactive peptide processing enzymes in Drosophila and provide the first evidence of two distinct glutaminyl cyclase isoforms located within one organism. Evolutionary studies revealed further links between bacterial aminopeptidases and the mammalian and insect glutaminyl cyclases. Previous work demonstrated a common fold and active site between human glutaminyl cyclase and bacterial aminopeptidase. Slight aminopeptidase activity was observed in the human glutaminyl cyclase and both glutaminyl cyclase isoforms. It was also determined the glutaminyl cyclases were able to bind leucine derivatives which are common aminopeptidase substrates. This observation led to the identification of a new class of competitive inhibitors for glutaminyl cyclases.

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