1jx0

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Chalcone Isomerase--Y106F mutant

Overview

In flavonoid, isoflavonoid, and anthocyanin biosynthesis, chalcone, isomerase (CHI) catalyzes the intramolecular cyclization of chalcones into, (S)-flavanones with a second-order rate constant that approaches the, diffusion-controlled limit. The three-dimensional structures of alfalfa, CHI complexed with different flavanones indicate that two sets of hydrogen, bonds may possess critical roles in catalysis. The first set of, interactions includes two conserved amino acids (Thr48 and Tyr106) that, mediate a hydrogen bond network with two active site water molecules. The, second set of hydrogen bonds occurs between the flavanone 7-hydroxyl group, and two active site residues (Asn113 and Thr190). Comparison of the, steady-state kinetic parameters of wild-type and mutant CHIs demonstrates, that efficient cyclization of various chalcones into their respective, flavanones requires both sets of contacts. For example, the T48A, T48S, Y106F, N113A, and T190A mutants exhibit 1550-, 3-, 30-, 7-, and 6-fold, reductions in k(cat) and 2-3-fold changes in K(m) with, 4,2',4'-trihydroxychalcone as a substrate. Kinetic comparisons of the, pH-dependence of the reactions catalyzed by wild-type and mutant enzymes, indicate that the active site hydrogen bonds contributed by these four, residues do not significantly alter the pK(a) of the intramolecular, cyclization reaction. Determinations of solvent kinetic isotope and, solvent viscosity effects for wild-type and mutant enzymes reveal a change, from a diffusion-controlled reaction to one limited by chemistry in the, T48A and Y106F mutants. The X-ray crystal structures of the T48A and Y106F, mutants support the assertion that the observed kinetic effects result, from the loss of key hydrogen bonds at the CHI active site. Our results, are consistent with a reaction mechanism for CHI in which Thr48 polarizes, the ketone of the substrate and Tyr106 stabilizes a key catalytic water, molecule. Hydrogen bonds contributed by Asn113 and Thr190 provide, additional stabilization in the transition state. Conservation of these, residues in CHIs from other plant species implies a common reaction, mechanism for enzyme-catalyzed flavanone formation in all plants.

About this Structure

1JX0 is a Single protein structure of sequence from Medicago sativa with SO4 and DFV as ligands. Active as Chalcone isomerase, with EC number 5.5.1.6 Full crystallographic information is available from OCA.

Reference

Role of hydrogen bonds in the reaction mechanism of chalcone isomerase., Jez JM, Bowman ME, Noel JP, Biochemistry. 2002 Apr 23;41(16):5168-76. PMID:11955065

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