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
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.