STEREOCHEMICAL AND ISOTOPIC LABELING STUDIES OF 2-OXO-HEPT-4-ENE-1,7-DIOATE HYDRATASE - EVIDENCE FOR AN ENZYME-CATALYZED KETONIZATION STEP IN THE HYDRATION REACTION

Citation
Ea. Burks et al., STEREOCHEMICAL AND ISOTOPIC LABELING STUDIES OF 2-OXO-HEPT-4-ENE-1,7-DIOATE HYDRATASE - EVIDENCE FOR AN ENZYME-CATALYZED KETONIZATION STEP IN THE HYDRATION REACTION, Journal of the American Chemical Society, 120(31), 1998, pp. 7665-7675
Citations number
50
Categorie Soggetti
Chemistry
ISSN journal
00027863
Volume
120
Issue
31
Year of publication
1998
Pages
7665 - 7675
Database
ISI
SICI code
0002-7863(1998)120:31<7665:SAILSO>2.0.ZU;2-1
Abstract
2-Oxo-hept-4-ene-1,7-dioate hydratase from Escherichia coli C converts 2-oxo-hept-4-ene-1,7-dioate to 2-oxo-4-hydroxy-hepta-1,7-dioate by th e addition of water using magnesium as a cofactor. The enzyme is one o f a set of inducible enzymes, known collectively as the homoprotocatec huate meta-fission pathway. The entire pathway enables the organism to utilize aromatic amino acids as its sole sources of carbon and energy . Expression and purification of 2-oxo-hept-4-ene-1,7-dioate hydratase to homogeneity permitted kinetic, isotopic labeling, and stereochemic al studies. Kinetic studies show that the enzyme processes either 2-ox o-hept-4-ene-1,7-dioate or 2-hydroxy-2,4-heptadiene-1,7-dioate to prod uct with comparable facility. Isotope labeling studies show that the h ydratase catalyzes the incorporation of a solvent deuteron at both C-3 and C-5 when the reaction is performed in (H2O)-H-2. The enzyme also accelerates the exchange of the C-3 proton of the alternate substrate 2-oxo-1,7-heptadioate with solvent deuterons. The results are consiste nt with a mechanism in which the enzyme catalyzes the isomerization of 2-oxo-hept-4-ene-1,7-dioate to its cr,P-unsaturated ketone followed b y the Michael addition of water. Whether this mechanistic sequence inv olves a one-base or a two-base mechanism is not yet known.