Leukotriene A4 hydrolase : identification of amino acid residues involved in catalyses and substrate-mediated inactivation

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.