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| | {{STRUCTURE_1jxz| PDB=1jxz | SCENE= }} | | {{STRUCTURE_1jxz| PDB=1jxz | SCENE= }} |
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| - | '''Structure of the H90Q mutant of 4-Chlorobenzoyl-Coenzyme A Dehalogenase complexed with 4-hydroxybenzoyl-Coenzyme A (product)'''
| + | ===Structure of the H90Q mutant of 4-Chlorobenzoyl-Coenzyme A Dehalogenase complexed with 4-hydroxybenzoyl-Coenzyme A (product)=== |
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| - | ==Overview==
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| - | 4-chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolytic dehalogenation of 4-CBA-CoA by attack of Asp145 on the C4 of the substrate benzoyl ring to form a Meisenheimer intermediate (EMc), followed by expulsion of chloride ion to form an arylated enzyme intermediate (EAr) and, finally, ester hydrolysis in EAr to form 4-hydroxybenzoyl-CoA (4-HBA-CoA). This study examines the contribution of the active site His90 to catalysis of this reaction pathway. The His90 residue was replaced with glutamine by site-directed mutagenesis. X-ray crystallographic analysis of H90Q dehalogenase complexed with 4-HBA-CoA revealed that the positions of the catalytic groups are unchanged from those observed in the structure of the 4-HBA-CoA-wild-type dehalogenase complex. The one exception is the Gln90 side chain, which is rotated away from the position of the His90 side chain. The vacated His90 site is occupied by two water molecules. Kinetic techniques were used to evaluate ligand binding and catalytic turnover rates in the wild-type and H90Q mutant dehalogenases. The rate constants for 4-CBA-CoA (both 7 microM(-1) x s(-1)) and 4-HBA-CoA (33 and 11 microM(-1) x s(-1)) binding to the two dehalogenases are similar in value. For wild-type dehalogenase, the rate constant for a single turnover is 2.3 s(-1) while that for multiple turnovers is 0.7 s(-1). For H90Q dehalogenase, these rate constants are 1.6 x 10(-2) and 2 x 10(-4) s(-1). The rate constants for EMc formation in wild-type and mutant dehalogenase are approximately 200 s(-1) while the rate constants for EAr formation are 40 and 0.3 s(-1), respectively. The rate constant for hydrolysis of EAr in wild-type dehalogenase is 20 s(-1) and in the H90Q mutant, 0.13 s(-1). The 133-fold reduction in the rate of EAr formation in the mutant may be the result of active site hydration, while the 154-fold reduction in the rate EAr hydrolysis may be the result of lost general base catalysis. Substitution of the His90 with Gln also introduces a rate-limiting step which follows catalysis, and may involve renewing the catalytic site through a slow conformational change.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_11695894}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 11695894 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_11695894}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Coenzyme-a]] | | [[Category: Coenzyme-a]] |
| | [[Category: Dehalogenase]] | | [[Category: Dehalogenase]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May 2 22:03:40 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jul 29 02:17:41 2008'' |
Revision as of 23:17, 28 July 2008
Template:STRUCTURE 1jxz
Structure of the H90Q mutant of 4-Chlorobenzoyl-Coenzyme A Dehalogenase complexed with 4-hydroxybenzoyl-Coenzyme A (product)
Template:ABSTRACT PUBMED 11695894
About this Structure
1JXZ is a Single protein structure of sequence from Pseudomonas sp.. Full crystallographic information is available from OCA.
Reference
Histidine 90 function in 4-chlorobenzoyl-coenzyme a dehalogenase catalysis., Zhang W, Wei Y, Luo L, Taylor KL, Yang G, Dunaway-Mariano D, Benning MM, Holden HM, Biochemistry. 2001 Nov 13;40(45):13474-82. PMID:11695894
Page seeded by OCA on Tue Jul 29 02:17:41 2008
