Title

The Mechanism of Pyroglutamate Formation

Date of Award

2002

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry and Biochemistry

First Advisor

Robert C. Bateman, Jr.

Advisor Department

Chemistry and Biochemistry

Abstract

Pyroglutamate is found at the N-terminus of several peptides and proteins in plants and mammals. This residue results from the cyclization of a glutaminyl residue, a reaction that in vivo , is catalyzed by glutaminyl cyclase (QC). To understand the mechanism of the enzymatic reaction, the nonenzymatic reactions involving the cyclization of glutamine, glutaminamide, and γ-glutamyl-p-nitroanilide were investigated. The nonenzymatic reaction rate depended on type of buffer, buffer concentration, pH, and temperature. The rate constants and activation parameters were determined. The nonenzymatic rate constants were then used to determine the catalytic proficiencies of QC. A mechanism involving the role of phosphate was proposed. QC is structurally related to the zinc aminopeptidase from A. proteolytica (AAP). Metal analysis of QC indicated that it is not a metalloenzyme. It did not exhibit aminopeptidase activity nor did the apoenzyme of AAP exhibit QC activity. AAP and QC have similar residues in their active sites. Kinetic analysis of some of these residues was performed in this study. His 307, and His 319 appeared to be involved in substrate binding while Asp 159 and Asp 248 do not appear to play a significant role in substrate binding or enzyme catalysis. On the basis of the nonenzymatic reaction, a mechanism involving the essential residues was proposed. Inhibition studies indicated that mammalian QC was inhibited by imidazole compounds, and 1,10-phenanthroline in a competitive manner. The most potent inhibitor of mammalian QC was 1,10-phenanthroline. Inhibition of imidazole compounds depended on the type and position of substituents on the imidazole ring. On the basis of this study, histamine compounds were used to prepare inhibitor columns by coupling them to carbodiimide-activated or epoxy-activated matrices. Preliminary experiments indicated that histamine, and 3-methylhistamine can be used to purify recombinant hQC. Papaya QC is not inhibited by imidazole compounds and 1,10-phenanthroline. Although papaya QC and mammalian QC differ in biochemical properties, substrate specificity, inhibitor recognition and secondary structure, they appear to adopt the same mechanism involving an intramolecular cyclization leading to the formation of a five membered intermediate.