Enzyme-catalyzed acylation of homoserine: Mechanistic characterization of the Haemophilus influenzae met2-encoded homoserine transacetylase

Citation
Tl. Born et al., Enzyme-catalyzed acylation of homoserine: Mechanistic characterization of the Haemophilus influenzae met2-encoded homoserine transacetylase, BIOCHEM, 39(29), 2000, pp. 8556-8564
Citations number
41
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
0006-2960 → ACNP
Volume
39
Issue
29
Year of publication
2000
Pages
8556 - 8564
Database
ISI
SICI code
0006-2960(20000725)39:29<8556:EAOHMC>2.0.ZU;2-V
Abstract
The first unique step in bacterial and plant methionine biosynthesis involv es the acylation of the gamma-hydroxyl of homoserine. In Haemophilus influe nzae, acylation is accomplished via an acetyl-CoA-dependent acetylation cat alyzed by homoserine transacetylase. The activity of this enzyme regulates flux of homoserine into multiple biosynthetic pathways and, therefore, repr esents a critical control point for cell growth and viability. We have clon ed homoserine transacetylase from PI. influenzae and present the first deta iled enzymatic study of this enzyme. Steady-state kinetic experiments demon strate that the enzyme utilizes a ping-pong kinetic mechanism in which the acetyl group of acetyl-CoA is initially transferred to an enzyme nucleophil e before subsequent transfer to homoserine to form the final product, O-ace tylhomoserine. The maximal velocity and V/K-homoserine were independent of pH over the range of values tested, while V/Kacetyl-CoA was dependent upon the ionization state of a single group exhibiting a pK value of 8.6, which was required to be protonated. Solvent kinetic isotope effect studies yield ed inverse effects of 0.75 on V and 0.74 on V/K-CoA on the reverse reaction and effects of 1.2 on V and 1.7 on V/K-homoserine on the forward reaction. Direct evidence for the formation of an acetyl-enzyme intermediate was obt ained using rapid-quench labeling studies. On the basis of these observatio ns, we propose a chemical mechanism for this important member of the acyltr ansferase family and contrast its mechanism with that of homoserine transsu ccinylase.