Title

The Identification of Essential Histidine Residues In Human Glutaminyl Cyclase

Date of Award

1999

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

Small biologically active peptides, peptide hormones, and neurotransmitters are synthesized as larger inactive precursor proteins that must be modified via posttranslational cleavage and enzymatic conversions to attain biological activity. One such modification is the masking of the N-terminus by cyclizing glutamine to pyroglutamic acid (pGlu). Glutaminyl cyclase (EC 2.3.2.5; glutamine cyclotransferase; QC) is the enzyme responsible for such modifications which usually renders peptides more resistant to degradation. To date, QC has been reported in a number of animals, bacteria, and plants. The cDNA for human QC was cloned into the GST expression vector which produced a soluble fusion protein consisting of the 27 kDa GST domain and the 38 kDa QC domain when expressed in E. coli . The enzyme had a specific activity of 1 μmole/min/mg with Gln-NH2 as a substrate, and a Km of 1.2 ± 0.02 mM at pH 7.8. The enzyme has a pH optimum of 7.5 with two pKa values of 6.6 and 9.1. Human QC was also found to by competitively inhibited by imidazole with a K I of 0.56 ± 0.03 mM at pH 7.8. Recombinant human QC was rapidly inactivated by diethylpyrocarbonate under neutral conditions. The pH dependence of DEPC inactivation revealed the modification of an essential amino acid residue with a pKa of 6.8 suggesting the involvement of histidine residues at the active site. DEPC inactivated QC exhibited a strong increase in absorbance at 240 nm and could be reactivated with 2 mM hydroxylamine, both signifying histidine involvement at the active site. Using Touchdown PCP, 75% of the coding region of sheep, pig, dog, rat, and chicken QC cDNA was amplified. Comparison of the amino acid sequences revealed a remarkably high identity of 81% among these species. Using these sequences to search GenBank, four sequences believed to be QC were found in Sacharomyces cerevisiae (yeast), Drosophila melanogaster (fruit fly), Caenorhabditis elegans (flat worm), and Bothrops jararaca (snake venom). Comparison of these sequences with human QC demonstrated the presence of four totally conserved histidine residues which were the targets for site directed mutagenesis. Stoichiometric studies of human QC suggested the presence of three histidine residues important for enzymatic activity. The conserved histidine residues mentioned above, at positions 140, 307, 319, and 330, were changed to glutamine residues. The resulting specific activities of the histidine mutants, when expressed in E. coli , revealed that His140 had no discernible specific activity compared to 632 ± 52.18 nmol/min/mg for native human QC. Also, while His307 retained 84% of the native QC activity, His319 retained only 35% and His330 12%. A 1,029 base pair fragment believed to be yeast QC was amplified from yeast genomic DNA. This PCR fragment was cloned into the pTrcHis2 TOPO vector. Sequencing of positive clones indicated that gene rearrangement had occurred during the cloning process. This was shown multiple times and is believed that the gene may be lethal to E. coli . Successful expression was not achieved.