1ghd

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{{STRUCTURE_1ghd| PDB=1ghd | SCENE= }}
{{STRUCTURE_1ghd| PDB=1ghd | SCENE= }}
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'''Crystal structure of the glutaryl-7-aminocephalosporanic acid acylase by mad phasing'''
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===Crystal structure of the glutaryl-7-aminocephalosporanic acid acylase by mad phasing===
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==Overview==
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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.
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{{ABSTRACT_PUBMED_11782466}}
==About this Structure==
==About this Structure==
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[[Category: Zhao, G.]]
[[Category: Zhao, G.]]
[[Category: Cephalosporin acylase]]
[[Category: Cephalosporin acylase]]
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''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jul 1 05:17:16 2008''

Revision as of 02:17, 1 July 2008

Image:1ghd.png

Template:STRUCTURE 1ghd

Crystal structure of the glutaryl-7-aminocephalosporanic acid acylase by mad phasing

Template:ABSTRACT PUBMED 11782466

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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