Pyroglutamyl Peptidase I (PAP1, EC 3.4.19.3) hydrolytically cleaves pyroglutamic acid (pGlu) from the N-terminal of most pGlu-peptides. In higher organisms Thyrothropin Releasing Hormone is a notable biologically active substrate of PAP1. The sequence of human PAP1 was obtained from GenBank at NCBI (www.ncbi.nlm.nih.gov). Using suitable primers cDNA was synthesised using RNA isolated from a human cell line. Functionally active recombinant human PAP1 was expressed in Escherichia coli. To facilitate both high expression levels and ease of purification a C-terminal His-tag fusion was made. It was shown that this fusion did not affect enzymatic activity. Yields of up to 25 mg purified recombinant human PAP1 per litre of culture were achieved. Gel filtration chromatography and SDS-PAGE data showed that the recombinant human PAP 1 exists as an active monomer. The Michaelis-Menten constant (KJ for the fluorometric substrate pGlu-7-amino-4-methyl coumarin was determined as 51.8 juM and the turnover constant (Kcat) was determined as 3.75 s'1. The activity of the enzyme displayed an absolute requirement for a thiol-reducing agent such as DTT. Optimal enzyme activity was observed at pH range 8.0-9.5 and temperature range 30-50 °C. The inhibitory effect of a range of compounds (e.g. 2-pyrrolidone) was measured. The three dimensional structure of human PAP1 was modelled on existing homologous PAP1 structures. Several amino acid residues were selected for site-directed mutagenesis. Kinetic analysis of the mutant PAP1 enzymes gave insight into the role these residues play within catalytic site, substrate binding pocket and other significant regions of PAP1. Preliminary determination of conditions under which recombinant human PAP1 forms protein crystals was carried out.