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| - | [[Image:2anp.gif|left|200px]] | + | {{Seed}} |
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| | {{STRUCTURE_2anp| PDB=2anp | SCENE= }} | | {{STRUCTURE_2anp| PDB=2anp | SCENE= }} |
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| - | '''Functional Glutamate 151 to Histidine mutant of the aminopeptidase from Aeromonas Proteolytica.'''
| + | ===Functional Glutamate 151 to Histidine mutant of the aminopeptidase from Aeromonas Proteolytica.=== |
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| - | ==Overview==
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| - | Glutamate151 (E151) has been shown to be catalytically essential for the aminopeptidase from Vibrio proteolyticus (AAP). E151 acts as the general acid/base during the catalytic mechanism of peptide hydrolysis. However, a glutamate residue is not the only residue capable of functioning as a general acid/base during catalysis for dinuclear metallohydrolases. Recent crystallographic characterization of the D-aminopeptidase from Bacillus subtilis (DppA) revealed a histidine residue that resides in an identical position to E151 in AAP. Because the active-site ligands for DppA and AAP are identical, AAP has been used as a model enzyme to understand the mechanistic role of H115 in DppA. Substitution of E151 with histidine resulted in an active AAP enzyme exhibiting a kcat value of 2.0 min(-1), which is over 2000 times slower than r AAP (4380 min(-1)). ITC experiments revealed that ZnII binds 330 and 3 times more weakly to E151H-AAP compared to r-AAP. UV-vis and EPR spectra of CoII-loaded E151H-AAP indicated that the first metal ion resides in a hexacoordinate/pentacoordinate equilibrium environment, whereas the second metal ion is six-coordinate. pH dependence of the kinetic parameters kcat and K(m) for the hydrolysis of L-leucine p-nitroanilide (L-pNA) revealed a change in an ionization constant in the enzyme-substrate complex from 5.3 in r-AAP to 6.4 in E151H-AAP, consistent with E151 in AAP being the active-site general acid/base. Proton inventory studies at pH 8.50 indicate the transfer of one proton in the rate-limiting step of the reaction. Moreover, the X-ray crystal structure of [ZnZn(E151H-AAP)] has been solved to 1.9 A resolution, and alteration of E151 to histidine does not introduce any major conformational changes to the overall protein structure or the dinuclear ZnII active site. Therefore, a histidine residue can function as the general acid/base in hydrolysis reactions of peptides and, through analogy of the role of E151 in AAP, H115 in DppA likely shuttles a proton to the leaving group of the substrate.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_16142900}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 16142900 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_16142900}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Spectroscopy]] | | [[Category: Spectroscopy]] |
| | [[Category: Zinc]] | | [[Category: Zinc]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sat May 3 19:15:43 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Mon Jul 28 18:15:41 2008'' |
Revision as of 15:15, 28 July 2008
Template:STRUCTURE 2anp
Functional Glutamate 151 to Histidine mutant of the aminopeptidase from Aeromonas Proteolytica.
Template:ABSTRACT PUBMED 16142900
About this Structure
2ANP is a Single protein structure of sequence from Vibrio proteolyticus. Full crystallographic information is available from OCA.
Reference
Kinetic, spectroscopic, and X-ray crystallographic characterization of the functional E151H aminopeptidase from Aeromonas proteolytica., Bzymek KP, Moulin A, Swierczek SI, Ringe D, Petsko GA, Bennett B, Holz RC, Biochemistry. 2005 Sep 13;44(36):12030-40. PMID:16142900
Page seeded by OCA on Mon Jul 28 18:15:41 2008
