1ghd
From Proteopedia
(New page: 200px<br /><applet load="1ghd" size="450" color="white" frame="true" align="right" spinBox="true" caption="1ghd, resolution 2.40Å" /> '''Crystal structure of...) |
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| - | [[Image:1ghd.gif|left|200px]]<br /><applet load="1ghd" size=" | + | [[Image:1ghd.gif|left|200px]]<br /><applet load="1ghd" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1ghd, resolution 2.40Å" /> | caption="1ghd, resolution 2.40Å" /> | ||
'''Crystal structure of the glutaryl-7-aminocephalosporanic acid acylase by mad phasing'''<br /> | '''Crystal structure of the glutaryl-7-aminocephalosporanic acid acylase by mad phasing'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130 (C130) | + | Glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130 (C130) was irreversibly inhibited in a time-dependent manner by two substrate analogs bearing side chains of variable length, namely 7beta-bromoacetyl aminocephalosporanic acid (BA-7-ACA) and 7beta-3-bromopropionyl aminocephalosporanic acid (BP-7-ACA). The inhibition of the enzyme with BA-7-ACA was attributable to reaction with a single amino acid residue within the beta-subunit proven by comparative matrix assisted laser desorption/ionization-time of flight mass spectrometry. Further mass spectrometric analysis demonstrated that the fourth tryptophan residue of the beta-subunit, Trp-B4, was alkylated by BA-7-ACA. By (1)H-(13)C HSQC spectroscopy of C130 labeled by BA-2-(13)C-7-ACA, it was shown that tryptophan residue(s) in the enzyme was alkylated, forming a carbon-carbon bond. Replacing Trp-B4 with other amino acid residues caused increases in K(m), decreases in k(cat), and instability of enzyme activity. None of the mutant enzymes except W-B4Y could be affinity-alkylated, but all were competitively inhibited by BA-7-ACA. Kinetic studies revealed that both BA-7-ACA and BP-7-ACA could specifically alkylate Trp-B4 of C130 as well as Tyr-B4 of the mutant W-B4Y. Because these alkylations were energy-requiring under physiological conditions, it is likely that the affinity labeling reactions were catalyzed by the C130 enzyme itself. The Trp-B4 residue is located in the middle of a characteristic alphabetabetaalpha sandwich structure. Therefore, a large conformational alteration during inhibitor binding and transition state formation is likely and suggests that a major conformational change is induced by substrate binding during the course of catalysis. |
==About this Structure== | ==About this Structure== | ||
| - | 1GHD is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Pseudomonas_sp._130 Pseudomonas sp. 130]. Full crystallographic information is available from [http:// | + | 1GHD is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Pseudomonas_sp._130 Pseudomonas sp. 130]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1GHD OCA]. |
==Reference== | ==Reference== | ||
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[[Category: cephalosporin acylase]] | [[Category: cephalosporin acylase]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:50:04 2008'' |
Revision as of 10:50, 21 February 2008
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Crystal structure of the glutaryl-7-aminocephalosporanic acid acylase by mad phasing
Overview
Glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130 (C130) was irreversibly inhibited in a time-dependent manner by two substrate analogs bearing side chains of variable length, namely 7beta-bromoacetyl aminocephalosporanic acid (BA-7-ACA) and 7beta-3-bromopropionyl aminocephalosporanic acid (BP-7-ACA). The inhibition of the enzyme with BA-7-ACA was attributable to reaction with a single amino acid residue within the beta-subunit proven by comparative matrix assisted laser desorption/ionization-time of flight mass spectrometry. Further mass spectrometric analysis demonstrated that the fourth tryptophan residue of the beta-subunit, Trp-B4, was alkylated by BA-7-ACA. By (1)H-(13)C HSQC spectroscopy of C130 labeled by BA-2-(13)C-7-ACA, it was shown that tryptophan residue(s) in the enzyme was alkylated, forming a carbon-carbon bond. Replacing Trp-B4 with other amino acid residues caused increases in K(m), decreases in k(cat), and instability of enzyme activity. None of the mutant enzymes except W-B4Y could be affinity-alkylated, but all were competitively inhibited by BA-7-ACA. Kinetic studies revealed that both BA-7-ACA and BP-7-ACA could specifically alkylate Trp-B4 of C130 as well as Tyr-B4 of the mutant W-B4Y. Because these alkylations were energy-requiring under physiological conditions, it is likely that the affinity labeling reactions were catalyzed by the C130 enzyme itself. The Trp-B4 residue is located in the middle of a characteristic alphabetabetaalpha sandwich structure. Therefore, a large conformational alteration during inhibitor binding and transition state formation is likely and suggests that a major conformational change is induced by substrate binding during the course of catalysis.
About this Structure
1GHD is a Protein complex structure of sequences from Pseudomonas sp. 130. Full crystallographic information is available from OCA.
Reference
Affinity alkylation of the Trp-B4 residue of the beta -subunit of the glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130., Huang X, Zeng R, Ding X, Mao X, Ding Y, Rao Z, Xie Y, Jiang W, Zhao G, J Biol Chem. 2002 Mar 22;277(12):10256-64. Epub 2002 Jan 8. PMID:11782466
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Categories: Protein complex | Pseudomonas sp. 130 | Bartlam, M. | Ding, Y. | He, H. | Jiang, F. | Jiang, W. | Liu, Y. | Mao, X. | Rao, Z. | Tang, H. | Ye, S. | Zhang, S. | Zhao, G. | Cephalosporin acylase
