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Homoserine Kinase
Background:
Homoserine kinase,
a key enzyme in the aspartate pathway of amino acid biosynthesis in Escherichia
coli, catalyzes the phosphorylation of L-homoserine to form
L-homoserine phosphate. The substrate of this reaction is also a precursor
for the branch of the pathway leading to the synthesis of the amino acid
methionine. The product is converted to threonine by threonine synthase and then eventually to isoleucine. The ThrB gene coding for this enzyme has been
cloned, and the enzyme has been overexpressed and purified to homogeneity
in high yield with a simplified purification scheme.
Results:
Spectral and
chemical characterization of the enzyme inactivated with several group
specific reagents has shown that activity loss is caused by the
modification of histidine and arginine residues. Treatment of the enzyme
with pyridoxal-5'-phosphate also results in enzyme inactivation. The
spectra evidence for the formation of a Schiff base, and the complete
protection afforded by substrates and inhibitors, indicate that homoserine kinase
also contains a lysine that is essential for catalytic activity (Huo & Viola, 1996). Site-directed mutagenesis
studies have confirmed a role for arginine-234 in the binding of the
carboxyl group of L-homoserine, and the involvement of two histidines at the homoserine binding site (Huo & Viola, 1996).
Mutations at these histidines have led to the
decoupling of the kinase activity from an inherent ATPase
activity in the enzyme, and suggest the presence of independent domains for
the binding of each substrate in homoserine kinase.
Kinetic Parameters of
Homoserine Kinase Mutants.
|
Enzyme
|
kcat
(sec-1) Kinase
|
kcat
(sec-1) ATPase
|
Kinase/ATPase Ratio
|
|
wild-type
|
18.3
|
0.016
|
1140
|
|
R234L
|
0.20
|
0.049
|
4.1
|
|
R234C
|
0.022
|
0.28
|
0.08
|
|
R234H
|
0.073
|
0.37
|
0.19
|
|
H139L
|
0.52
|
2.51
|
0.21
|
|
H202L
|
9.06
|
0.071
|
128
|
|
H205Q
|
0.005
|
0.036
|
0.14
|
|
