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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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Published in: | PloS one 2018-01, Vol.13 (1), p.e0190816-e0190816 |
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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. |
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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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0190816</identifier><identifier>PMID: 29351301</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Alanine ; Aminopeptidase ; Aminopeptidases ; Analysis ; Angiotensin ; Binding sites ; Biology and Life Sciences ; Bradykinin ; Catabolism ; Catalysis ; Chemistry ; Cloning ; Deoxyribonucleic acid ; Dimers ; DNA ; DNA polymerase ; E coli ; Enzymatic activity ; Enzyme kinetics ; Genetic aspects ; Guanidine hydrochloride ; Hydrogen ; Hydrogen bonds ; Inflammation ; Investigations ; Kinins ; Medicine and Health Sciences ; Metalloproteinase ; Mutagenesis ; Mutants ; Peptides ; Physical Sciences ; Physiological aspects ; Proline ; Protein structure ; Proteins ; Protons ; Quaternary structure ; Relay systems ; Research and Analysis Methods ; Restoration ; Solvents ; Substrates ; Turnover rate ; Ultracentrifugation ; Vasoactive agents</subject><ispartof>PloS one, 2018-01, Vol.13 (1), p.e0190816-e0190816</ispartof><rights>COPYRIGHT 2018 Public Library of Science</rights><rights>2018 Chang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2018 Chang et al 2018 Chang et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-6ea5c5608d2a34a2e1a40080a234f9433612df72e3e434a6e409ea995e4cd7283</citedby><cites>FETCH-LOGICAL-c692t-6ea5c5608d2a34a2e1a40080a234f9433612df72e3e434a6e409ea995e4cd7283</cites><orcidid>0000-0003-3210-0543</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.proquest.com/docview/2390623753/fulltextPDF?pq-origsite=primo$$EPDF$$P50$$Gproquest$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.proquest.com/docview/2390623753?pq-origsite=primo$$EHTML$$P50$$Gproquest$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,885,25753,27924,27925,37012,37013,44590,53791,53793,75126</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/29351301$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Silman, Israel</contributor><creatorcontrib>Chang, Hui-Chuan</creatorcontrib><creatorcontrib>Kung, Camy C-H</creatorcontrib><creatorcontrib>Chang, Tzu-Ting</creatorcontrib><creatorcontrib>Jao, Shu-Chuan</creatorcontrib><creatorcontrib>Hsu, Yu-Ting</creatorcontrib><creatorcontrib>Li, Wen-Shan</creatorcontrib><title>Investigation of the proton relay system operative in human cystosolic aminopeptidase P</title><title>PloS one</title><addtitle>PLoS One</addtitle><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.</description><subject>Alanine</subject><subject>Aminopeptidase</subject><subject>Aminopeptidases</subject><subject>Analysis</subject><subject>Angiotensin</subject><subject>Binding sites</subject><subject>Biology and Life Sciences</subject><subject>Bradykinin</subject><subject>Catabolism</subject><subject>Catalysis</subject><subject>Chemistry</subject><subject>Cloning</subject><subject>Deoxyribonucleic acid</subject><subject>Dimers</subject><subject>DNA</subject><subject>DNA polymerase</subject><subject>E coli</subject><subject>Enzymatic activity</subject><subject>Enzyme kinetics</subject><subject>Genetic aspects</subject><subject>Guanidine hydrochloride</subject><subject>Hydrogen</subject><subject>Hydrogen bonds</subject><subject>Inflammation</subject><subject>Investigations</subject><subject>Kinins</subject><subject>Medicine and Health Sciences</subject><subject>Metalloproteinase</subject><subject>Mutagenesis</subject><subject>Mutants</subject><subject>Peptides</subject><subject>Physical Sciences</subject><subject>Physiological aspects</subject><subject>Proline</subject><subject>Protein structure</subject><subject>Proteins</subject><subject>Protons</subject><subject>Quaternary structure</subject><subject>Relay systems</subject><subject>Research and Analysis Methods</subject><subject>Restoration</subject><subject>Solvents</subject><subject>Substrates</subject><subject>Turnover rate</subject><subject>Ultracentrifugation</subject><subject>Vasoactive 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of the proton relay system operative in human cystosolic aminopeptidase P</title><author>Chang, Hui-Chuan ; Kung, Camy C-H ; Chang, Tzu-Ting ; Jao, Shu-Chuan ; Hsu, Yu-Ting ; Li, Wen-Shan</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-6ea5c5608d2a34a2e1a40080a234f9433612df72e3e434a6e409ea995e4cd7283</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2018</creationdate><topic>Alanine</topic><topic>Aminopeptidase</topic><topic>Aminopeptidases</topic><topic>Analysis</topic><topic>Angiotensin</topic><topic>Binding sites</topic><topic>Biology and Life Sciences</topic><topic>Bradykinin</topic><topic>Catabolism</topic><topic>Catalysis</topic><topic>Chemistry</topic><topic>Cloning</topic><topic>Deoxyribonucleic acid</topic><topic>Dimers</topic><topic>DNA</topic><topic>DNA polymerase</topic><topic>E coli</topic><topic>Enzymatic activity</topic><topic>Enzyme kinetics</topic><topic>Genetic aspects</topic><topic>Guanidine hydrochloride</topic><topic>Hydrogen</topic><topic>Hydrogen bonds</topic><topic>Inflammation</topic><topic>Investigations</topic><topic>Kinins</topic><topic>Medicine and Health Sciences</topic><topic>Metalloproteinase</topic><topic>Mutagenesis</topic><topic>Mutants</topic><topic>Peptides</topic><topic>Physical Sciences</topic><topic>Physiological aspects</topic><topic>Proline</topic><topic>Protein structure</topic><topic>Proteins</topic><topic>Protons</topic><topic>Quaternary structure</topic><topic>Relay systems</topic><topic>Research and Analysis Methods</topic><topic>Restoration</topic><topic>Solvents</topic><topic>Substrates</topic><topic>Turnover rate</topic><topic>Ultracentrifugation</topic><topic>Vasoactive agents</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Chang, Hui-Chuan</creatorcontrib><creatorcontrib>Kung, Camy C-H</creatorcontrib><creatorcontrib>Chang, 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Israel</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Investigation of the proton relay system operative in human cystosolic aminopeptidase P</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2018-01-19</date><risdate>2018</risdate><volume>13</volume><issue>1</issue><spage>e0190816</spage><epage>e0190816</epage><pages>e0190816-e0190816</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>29351301</pmid><doi>10.1371/journal.pone.0190816</doi><tpages>e0190816</tpages><orcidid>https://orcid.org/0000-0003-3210-0543</orcidid><oa>free_for_read</oa></addata></record> |
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subjects | Alanine Aminopeptidase Aminopeptidases Analysis Angiotensin Binding sites Biology and Life Sciences Bradykinin Catabolism Catalysis Chemistry Cloning Deoxyribonucleic acid Dimers DNA DNA polymerase E coli Enzymatic activity Enzyme kinetics Genetic aspects Guanidine hydrochloride Hydrogen Hydrogen bonds Inflammation Investigations Kinins Medicine and Health Sciences Metalloproteinase Mutagenesis Mutants Peptides Physical Sciences Physiological aspects Proline Protein structure Proteins Protons Quaternary structure Relay systems Research and Analysis Methods Restoration Solvents Substrates Turnover rate Ultracentrifugation Vasoactive agents |
title | Investigation of the proton relay system operative in human cystosolic aminopeptidase P |
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