Access Type

Open Access Thesis

Date of Award

January 2014

Degree Type

Thesis

Degree Name

M.S.

Department

Pharmaceutical Sciences

First Advisor

Steven M. Firestine

Abstract

The de novo purine biosynthesis pathway plays a critical role in providing new purines to the cell. Previous studies have shown differences between the human and bacterial pathways which suggests that pathway may be a potential target for antibiotic drug discovery. Three critical enzymes are involved in the pathway divergence. In the bacterial pathway, N5-CAIR synthetase (PurK) first converts AIR to N5-CAIR, which is then transformed into CAIR catalyzed by the enzyme N5-CAIR mutase (Class I PurE). In the human pathway, AIR carboxylase (Class II PurE) catalyzes the direct conversion of AIR to CAIR. Class I and Class II PurEs have structure and sequence similarities but also are functionally different. However, the residues responsible for these differences are unknown. In this study, we hypnotized that the class-specific conserved residues in the active site might be the key to determining the functional differences between the two PurEs. Site-directed mutagenesis was used to convert residues of Class I PurEs to Class II PurEs. Several mutant PurEs were made and examined for CO2-dependent conversion of AIR to CAIR. Two mutants H71A and H71G displayed CO2 dependent that was similar to that observed for AIR carboxylase. Additional studies revealed that these enzymes also were capable of using N5-CAIR as a substrate. This indicates that the H71A and H71G mutant have AIR carboxylase as well as N5-CAIR mutase activity. Examination of the crystal structure of N5-CAIR mutase indicates that His71 is at the bottom of the active site and removal of this residue creates a channel between active sites in two subunits. We speculate that this channel may be key for the utilization of CO2.

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