Escherichia coli IGP synthase: Subunit communication in a glutamine amidotransferase

Thomas John Klem, Purdue University

Abstract

Glutamine amidotransferases (GATs) incorporate the amide nitrogen of glutamine onto a variety of primary metabolites. These enzymes contain functional glutamine binding and "synthase" domains that coordinate their actions to ensure incorporation of nitrogen onto the substrate. GATs have three common catalytic properties: (1) the ability to use ammonia as a nitrogen source, (2) the ability to hydrolyze the amide nitrogen group from glutamine (glutaminase activity), and (3) irreversible inactivation by glutamine analogs. Escherichia coli IGP synthase is required for histidine biosynthesis. It is a heterodimeric enzyme, consisting of the HisH (glutamine binding) and HisF (synthase) subunits. In the process of characterizing the enzyme, unusual properties with respect to the features described above were discovered: the HisH subunit did not exhibit glutaminase activity unless it was associated with HisF; however, HisH could be modified by glutamine analogs in the absence of the synthase domain. These two observations provided the foundation for additional work directed toward understanding how the subunits interact during normal turnover. The HisH subunit is required for the enzyme to utilize glutamine; the lack of a detectable glutaminase in HisH implies that HisF "activates" the glutamine binding subunit. Therefore, mutations on HisF were sought that resulted in IGP synthases deficient in the ability to utilize glutamine. These mutants have been characterized, and the role of the HisF domain in amide nitrogen release and transfer are discussed. A number of glutamine analogs exist that are irreversible inhibitors of glutamine-utilizing enzymes. During the course of studies with the analog acivicin, unexpected kinetic properties were discovered with regard to the inactivation of IGP synthase. The behavior of acivicin under various conditions is described, and explanations for the behavior put forth in the context of subunit interaction during the normal turnover event.

Degree

Ph.D.

Advisors

Davisson, Purdue University.

Subject Area

Biochemistry

COinS