Catalytic mechanisms and evolution of leukotriene A4 hydrolyse

Abstract

Inflammation is the first response of the body to infection or physical irritation. This response can potentially trigger the whole immune system but in the initial stage mainly involves leukocytes of the innate immune system and a complex cascade of chemical mediators, which by different means control, maintain and resolve the process. One group of such mediators is the leukotrienes, among which LTB4 is found. LTB4 has several immunomodulating properties and mainly acts by recruiting leukocytes to the site of injury or infection. LTB4 is mainly formed by leukocytes of the innate immune system, but recruits cells of both the innate as well as the adaptive immune systems. Thus, it constitutes an important link between the two systems. Moreover, LTB4 is known to be involved in several pathological inflammatory conditions.

Leukotriene A4 hydrolase (LTA4H) is a bifunctional zinc metalloenzyme that catalyzes the last step in the formation of LTB4. In addition, LTA4H catalyzes hydrolysis of oligo-peptides. Hence the enzyme is bifunctional and the two activities of LTA4H actually share a common active site. While LTB4 has a characterized biological functions in the inflammatory response, the physiological relevance of the peptidase activity of LTA4H is yet unknown.

According to sequence homology, LTA4H sorts as a member of the M1 family of aminopeptidases, a vast enzyme family found in most organisms and with a variety of biological functions. Among these peptidases, a subset present in vertebrates has the intrinsic capacity to catalyze LTA4 ¨ LTB4 formation.

The present investigations deal with the catalytic mechanism of both activities of LTA4H, as well as the divergent evolution of LTA4H from ancestral aminopeptidases. For the first part of the work, the recently determined crystal structure of LTA4H served as a guideline for the design of experiments. In these, an approach mainly including mutagenesis, enzyme kinetics, molecular modeling and crystallography led to determination of the basic requirements for catalysis by LTA4H as well as an insight into the molecular events underlying its evolution from ancestral aminopeptidases. To refine these findings, a novel assay for enzyme kinetics was developed, which allowed a faster and more adequate analysis of the peptidase activity. Finally, the novel assay in combination with crystal structure determinations of LTA4H in complex with different ligands, allowed a yet deeper understanding of the reaction mechanism for peptide hydrolysis catalyzed by LTA4H.

For the peptidase activity it was specifically shown that the peptide substrate is anchored between Arg-563 and Glu-271 with its C- and N-terminal, respectively. Together, these two residues function as an effective filter which selectively favors peptide substrates consisting of three residues, i.e. tri-peptides. The specific preference of the enzyme for arginyl tripeptides is achieved by interaction between basic N-terminal groups of the substrate and Asp-375. During the course of peptide hydrolysis, Tyr-383 together with the zinc ion function as a site for oxyanion stabilization of the reaction intermediate. The general base Glu-296, not only facilitates the nucleophilic attack by the hydrolytic water, but also functions as a proton shuffle, which in the last step of peptide hydrolysis protonates the leaving arnine. Considering sequence homology, these findings to a large extent holds true also for other metallopeptidases of the MA clan and specifically for aminopeptidases of the M1 family.

For the epoxide hydrolase activity, it was shown that the carboxylate group of LTA4 binds to Arg-563 to achieve proper positioning, with respect to catalytic residues, of the reacting moieties of the substrate. During LTA4 hydrolysis, Glu-271 and Asp-375 are required for epoxide ring opening and for catalyzing the stereospecific attack by the hydrolytic water at carbon 12 of LTA4.

Notably, Arg-563 and Glu-271 are essential for both reaction mechanisms. While Arg-563 serves the same purpose in both reaction mechanisms, i.e. binding of substrate carboxylates, Glu-271 has distinct roles in each reaction. The latter observation, with a single residue serving in two different catalytic mechanisms, is unique to LTA4H.

Often, assaying peptidase activity either involves complex assays, when utilizing natural substrates, or relies on chrornogenic model substrates of limited physiological relevance. The novel assay developed circumvents these problems and allows simple and fast screening of natural peptide substrates and determination of kinetic constants for their hydrolysis.

For the evolutionary studies, the yeast homologue of human LTA4H, an aminopeptidase with substrate specificities distinct ftom, human LTA4H, was used. This enzyme is activated by LTA4 but also to some extent hydrolyzes it. For peptide hydrolysis, it was shown that the yeast enzyme uses the corresponding residues as, human LTA4H. Additionally, it was shown that the active site pocket of the yeast enzyme allows LTA4 to bind in two conformations: one peptidase-activating and one compatible with LTA4 hydrolysis. A few point mutations of the yeast enzyme, which made it more similar to human LTA4H, sufficed to reengineer the pocket to more resemble the corresponding human one with LTA4-inhibition replacing the LTA4-activating effect. Thus, it appears as LTA4H through evolution has fine-tuned an existing lipid binding site to optimize it for LTA4-binding and turnover.