Abstract:
Leukotriene (LT) A4 hydrolase catalyzes the committed step in the
biosynthesis of LTB4, a classical chemoattractant and immuine-modulating
lipid mediator involved in inflammation, host-defense against infections,
and systemic, PAF-mediated, lethal shock. LTA4 hydrolase is a
bifunctional zinc metalloenzyme with a chloride stimulated arginyl
aminopeptidase activity. When exposed to its lipid substrate LTA4, the
enzyme is inactivated and covalently modified in a process termed suicide
inactivation, which puts a restrain on the enzymes ability to form the
biologically active LTB4.
In the present thesis, chemical modification with a series of amino
acid-specific reagents, in the presence and absence of competitive
inhibitors, was used to identify catalytically important residues at the
active site. Thus, using differential labeling techniques, modification
with the tyrosyl reagents N- acetylimidazole and tetranitromethane
revealed the presence of two catalytically important Tyr residues.
Likewise, modification with 2,3-butanedione and phenylglyoxal indicated
that three Arg residues were located at, or near, the active center of
the enzyme.
Using differential Lys-specific peptide mapping of untreated and suicide
inactivated LTA4 hydrolase, a 21 residue peptide termed K21, was
identified that is involved in binding of LTA4 to the native protein.
Isolation and amino acid sequencing of a modified form of K21, revealed
that Tyr-378 is the site of attachment between LTA4 and the protein. To
investigate the functional role of Tyr- 378, this residue was subjected
to mutational analysis. Thus, Tyr-378 was exchanged for a Phe and Gln
residue and the purified recombinant enzymes were characterized. Upon
exposure to LTA4, none of the mutated enzymes were susceptible to
inactivation and covalent modification by LTA4. In addition, the most
conservative mutation generated an enzyme, [Y378F]LTA4hydrolase, that
exhibited an increased (2.5 fold) turnover of LTA4 into LTB4, presumably
due to a reduced catalytic restrain normally imposed by suicide
inactivation. Hence, by a single point mutation we generated an enzyme
that is protected from suicide inactivation and exhibits an increased
catalytic efficiency. Moreover, we had shown that catalysis and covalent
modification/inactivation could be completely dissociated.
Both mutants of Tyr-378 were able to convert LTA4 into a novel product
that was structurally identified by UV spectroscopy, GC/MS, UV-induced
double-bond isomerization, and comparisons with synthetic standards.
Thus, we could show that these two mutants could generate both LTB4 and
the double-bond isomer delta6-trans-delta8-cis-LTB4 The fact that
mutation of Tyr-378 allows formation of a double-bond isomer of the
natural product LTB4, suggests that this residue may be involved in the
alignment of the substrate, or a carbocation derived thereof, in the
active site.
To detail the molecular mechanism for substrate-mediated inactivation of
LTA4, further analysis of mutated enzymes with peptide mapping and enzyme
activity determinations was carried out. Thus, we exposed the
functionally incompetent mutant [E318A]LTA4 hydrolase for the substrate
LTA4, and found that this protein was covalently modified to the same, or
even higher, extent as the catalytically active wild type enzyme. Hence,
the catalytic machinery is not required to activate the substrate to a
molecular species that can bind to the protein. Together with other data
from our laboratory as well as the inherent chemical properties of LTA4,
we conclude that substrate- mediated inactivation of LTA4 hydrolase
follows an affinity labeling mechanism, rather than a mechanism-based
process. This conclusion predicts (i) that LTA4 is likely to destroy
other enzymes/protein in its neighborhood to which it binds with
sufficient strength and (ii) that LTA4 can not be used as a template for
design of a suicide-type inhibitors.
