Investigation of the proton relay system operative in human cystosolic aminopeptidase P.
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 inflamma...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2018-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC5774706?pdf=render |
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| author | Hui-Chuan Chang Camy C-H Kung Tzu-Ting Chang Shu-Chuan Jao Yu-Ting Hsu Wen-Shan Li |
| author_facet | Hui-Chuan Chang Camy C-H Kung Tzu-Ting Chang Shu-Chuan Jao Yu-Ting Hsu Wen-Shan Li |
| author_sort | Hui-Chuan Chang |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-21T17:10:05Z |
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| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-21T17:10:05Z |
| publishDate | 2018-01-01 |
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| spelling | doaj.art-a65cee0345d1458caaf358387348c82b2022-12-21T18:56:26ZengPublic Library of Science (PLoS)PLoS ONE1932-62032018-01-01131e019081610.1371/journal.pone.0190816Investigation of the proton relay system operative in human cystosolic aminopeptidase P.Hui-Chuan ChangCamy C-H KungTzu-Ting ChangShu-Chuan JaoYu-Ting HsuWen-Shan LiAminopeptidase 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.http://europepmc.org/articles/PMC5774706?pdf=render |
| spellingShingle | Hui-Chuan Chang Camy C-H Kung Tzu-Ting Chang Shu-Chuan Jao Yu-Ting Hsu Wen-Shan Li Investigation of the proton relay system operative in human cystosolic aminopeptidase P. PLoS ONE |
| title | Investigation of the proton relay system operative in human cystosolic aminopeptidase P. |
| title_full | Investigation of the proton relay system operative in human cystosolic aminopeptidase P. |
| title_fullStr | Investigation of the proton relay system operative in human cystosolic aminopeptidase P. |
| title_full_unstemmed | Investigation of the proton relay system operative in human cystosolic aminopeptidase P. |
| title_short | Investigation of the proton relay system operative in human cystosolic aminopeptidase P. |
| title_sort | investigation of the proton relay system operative in human cystosolic aminopeptidase p |
| url | http://europepmc.org/articles/PMC5774706?pdf=render |
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