Investigation of the proton relay system operative in human cystosolic aminopeptidase P
| Autor: | Yu-Ting Hsu, Wen-Shan Li, Shu-Chuan Jao, Hui-Chuan Chang, Camy C.H. Kung, Tzu-Ting Chang |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Models Molecular Protein Denaturation Dimer lcsh:Medicine Pathology and Laboratory Medicine Guanidines Biochemistry Physical Chemistry Aminopeptidases chemistry.chemical_compound Cytosol Enzyme Stability Medicine and Health Sciences Guanidine lcsh:Science Alanine chemistry.chemical_classification Escherichia Coli Multidisciplinary Physics Recombinant Proteins Bacterial Pathogens Enzymes Chemistry Experimental Organism Systems Medical Microbiology Physical Sciences Prokaryotic Models Protons Pathogens Research Article Escherichia Hydrochloride Stereochemistry Chemical physics Research and Analysis Methods Michaelis–Menten kinetics Microbiology Catalysis 03 medical and health sciences Model Organisms Enterobacteriaceae Humans Enzyme kinetics Amino Acid Sequence Protein Structure Quaternary Microbial Pathogens Nuclear Physics Nucleons 030102 biochemistry & molecular biology Bacteria Chemical Bonding Gut Bacteria lcsh:R Chemical Compounds Organisms Biology and Life Sciences Proteins Hydrogen Bonding Dimers (Chemical physics) Kinetics Enzyme chemistry Amino Acid Substitution Enzyme Structure Enzymology Mutagenesis Site-Directed Protein quaternary structure lcsh:Q Protein Multimerization |
| Zdroj: | PLoS ONE, Vol 13, Iss 1, p e0190816 (2018) PLoS ONE |
| ISSN: | 1932-6203 |
| Popis: | Aminopeptidase P, a metalloprotease, targets Xaa-Proline peptides for cleavage [1-4]. There are two forms of human AMPP, a membrane-bound form (hmAMPP) and a soluble cytosolic form (hcAMPP)[5]. Similar to the angiotensin-I-converting enzyme, AMPP plays an important role in the catabolism of inflammatory and vasoactive peptides, known as kinins. The plasma kinin, bradykinin, was used as the substrate to conduct enzymatic activity analyses and to determine the Michaelis constant (Km) of 174 μM and the catalytic rate constant (kcat) of 10.8 s-1 for hcAMPP. Significant differences were observed in the activities of Y527F and R535A hcAMPP mutants, which displayed a 6-fold and 13.5-fold for decrease in turnover rate, respectively. Guanidine hydrochloride restored the activity of R535A hcAMPP, increasing the kcat/Km 20-fold, yet it had no impact on the activities of the wild-type or Y527F mutant hcAMPPs. Activity restoration by guanidine derivatives followed the order guanidine hydrochloride >> methyl-guanidine > amino-guanidine > N-ethyl-guanidine. Overall, the results indicate the participation of R535 in the hydrogen bond network that forms a proton relay system. The quaternary structure of hcAMPP was determined by using analytical ultracentrifugation (AUC). The results show that alanine replacement of Arg535 destabilizes the hcAMPP dimer and that guanidine hydrochloride restores the native monomer-dimer equilibrium. It is proposed that Arg535 plays an important role in hcAMMP catalysis and in stabilization of the catalytically active dimeric state. |
| Databáze: | OpenAIRE |
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