A molecular and biochemical study of two recombinant mammalian pyroglutamyl peptidases type 1

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 bovine PAP1 (Accession No XM 866409) was obtained from GenBank at NCBI (www ncbi nlm mh gov). Using suitable primers cDNA was synthesised using RNA extracted from bovine brain tissue. Following expression of recombinant bovine PAP1 in Escherichia cob, the protein was purified using immobilised nickel affinity chromatography resulting in a yield o f 2 6 mg o f PAP1 per litre culture The Michaehs- Menten constant (KJ fo r the fluorometnc substrate pGlu-7-amino-4-methyl coumarin was determined as 59 fiM and the turnover constant (Kcat) was determined as 3 5 s1. Optimal enzyme activity was observed at pH range 9 0-9 5 and temperature range 30- 37 °C. A comparative study carried out with the human and bovine recombinant forms of the enzyme has highlighted interesting differences at ammo acid, expression and enzymatic activity levels. Site-directed mutagenesis of human PAP1 has revealed that an acidic residue is required for catalytic activity. A series of active mutants were generated for human PAP1 using a random mutagenesis approach. Biochemical and kinetic analysis of the mutant PAP1 enzymes has shown that methionine residues could potentially have an important role in PAP1 protein expression. An attempt to crystallise human PAP1 was carried out. However, this proved to be unsuccessful although it is believed that the C-terminal His-tag is causing interference and thus preventing proper crystallisation of human PAP1

Cloning and heterologous expression of bovine pyroglutamyl peptidase type-1 in Escherichia coli : purification , biochemical and kinetic characterisation

We describe the cloning, expression and purification of the bovine XM866409 form of pyroglutamyl-aminopeptidase I. The amino acid sequence, deduced from the nucleotide sequence, revealed that it consists of 209 amino acid residues and showed to have 98% homology with the human AJ278828 form of the enzyme. Three amino acid residues at positions 81, 205 and 208 were found to vary among the two sequences. The bovine enzyme was expressed in XL10-gold Esherichia coli cells. Immobilizied Ni-ion affinity chromatography was used to purify the expressed protein resulting in a yield of 3.3mg of PAP1 per litre culture. The purified enzyme had a specific activity of 1700 units/ml. SDS-PAGE produced a single band for bovine PAP1 with a molecular weight of ~23-24 kDa which is in good agreement with previously reported data on PAP1. Kinetic constants Km and Kcat were 59μΜ and 3.5s-1, respectively. It possessed an optimum pH between 9-9.5, a temperature of 37°C and showed an absolute requirement for a thiol-reducing agent (10mM DTT). EDTA didn’t prove to have an effect on enzyme activity. Competitive inhibition was seen with pyroglutamyl peptides pGlu-His-Pro-NH2 (TRH; Ki= 44.1 uM), pGlu-Ala- OH (Ki=141 uM) and pGlu-Val-OH (Ki=652.17)

A molecular and biochemical study of two recombinant mammalian pyroglutamyl peptidases type 1

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 bovine PAP1 (Accession No XM 866409) was obtained from GenBank at NCBI (www ncbi nlm mh gov). Using suitable primers cDNA was synthesised using RNA extracted from bovine brain tissue. Following expression of recombinant bovine PAP1 in Escherichia cob, the protein was purified using immobilised nickel affinity chromatography resulting in a yield o f 2 6 mg o f PAP1 per litre culture The Michaehs- Menten constant (KJ fo r the fluorometnc substrate pGlu-7-amino-4-methyl coumarin was determined as 59 fiM and the turnover constant (Kcat) was determined as 3 5 s1. Optimal enzyme activity was observed at pH range 9 0-9 5 and temperature range 30- 37 °C. A comparative study carried out with the human and bovine recombinant forms of the enzyme has highlighted interesting differences at ammo acid, expression and enzymatic activity levels. Site-directed mutagenesis of human PAP1 has revealed that an acidic residue is required for catalytic activity. A series of active mutants were generated for human PAP1 using a random mutagenesis approach. Biochemical and kinetic analysis of the mutant PAP1 enzymes has shown that methionine residues could potentially have an important role in PAP1 protein expression. An attempt to crystallise human PAP1 was carried out. However, this proved to be unsuccessful although it is believed that the C-terminal His-tag is causing interference and thus preventing proper crystallisation of human PAP1