1jxz

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spinqualitylabelsall models 1jxz, resolution 1.9Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Structure of the H90Q mutant of 4-Chlorobenzoyl-Coenzyme A Dehalogenase complexed with 4-hydroxybenzoyl-Coenzyme A (product)

Overview

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.

About this Structure

1JXZ is a Single protein structure of sequence from Pseudomonas sp. with CA, PO4 and BCA as ligands. Active as 4-chlorobenzoate dehalogenase, with EC number 3.8.1.6 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

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