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

Timothy L. Born, Matthew Franklin, John S. Blanchard

Research output: Contribution to journalArticle

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Abstract

The first unique step in bacterial and plant methionine biosynthesis involves the acylation of the γ-hydroxyl of homoserine. In Haemophilus influenzae, acylation is accomplished via an acetyl-CoA-dependent acetylation catalyzed by homoserine transacetylase. The activity of this enzyme regulates flux of homoserine into multiple biosynthetic pathways and, therefore, represents a critical control point for cell growth and viability. We have cloned homoserine transacetylase from H. influenzae and present the first detailed enzymatic study of this enzyme. Steady-state kinetic experiments demonstrate that the enzyme utilizes a ping-pong kinetic mechanism in which the acetyl group of acetyl-CoA is initially transferred to an enzyme nucleophile before subsequent transfer to homoserine to form the final product, O-acetylhomoserine. The maximal velocity and V/K(homoserine) were independent of pH over the range of values tested, while V/K(acetyl-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 yielded 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 obtained using rapid-quench labeling studies. On the basis of these observations, we propose a chemical mechanism for this important member of the acyltransferase family and contrast its mechanism with that of homoserine transsuccinylase.

Original languageEnglish (US)
Pages (from-to)8556-8564
Number of pages9
JournalBiochemistry
Volume39
Issue number29
DOIs
StatePublished - Jul 25 2000

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homoserine O-acetyltransferase
Homoserine
Acylation
Haemophilus influenzae
Acetyl Coenzyme A
Enzymes
Homoserine O-Succinyltransferase
Kinetics
Acyltransferases
Acetylation
Nucleophiles
Biosynthesis
Biosynthetic Pathways
Cell growth
Isotopes
Methionine
Hydroxyl Radical
Labeling
Ionization
Cell Survival

ASJC Scopus subject areas

  • Biochemistry

Cite this

Enzyme-catalyzed acylation of homoserine : Mechanistic characterization of the Haemophilus influenzae met2-encoded homoserine transacetylase. / Born, Timothy L.; Franklin, Matthew; Blanchard, John S.

In: Biochemistry, Vol. 39, No. 29, 25.07.2000, p. 8556-8564.

Research output: Contribution to journalArticle

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