Leukotriene A4 hydrolase : exploration of the active sites and catalytic mechanisms by site-directed mutagenesis

Abstract

Leukotriene (LT) A4 is a pivotal intermediate in the biosynthesis of leukotrienes, a family of potent lipid mediators involved in a variety of inflammatory and allergic disorders. The bifunctional zinc metalloenzyme LTA4 hydrolase converts LTA4 into LTB4, one of the most potent chemotaxins known to date. In addition, LTA4 hydrolase possesses a peptide cleaving activity, the physiological role of which is presently unknown. From sequence comparisons with aminopeptidase M, Tyr-383 in LTA4 hydrolase was suggested as a potential catalytic residue. Tyr-383 was exchanged to a Phe, His or Gln by site-directed mutagenesis, and the purified recombinant enzymes were devoid of peptidase activity but displayed significant epoxide hydrolase activity.

The results indicate that Tyr-383 is located at the active site, and suggests a role for Tyr-383 in the peptidase reaction where it may serve as a proton donor. Mutagenetic replacement of the conserved Glu-296 in LTA4 hydrolase with a Gln, selectively abrogates its peptidase activity. Similar results were obtained when Glu was exchanged for Asp or Asn. Thus, a carboxylate at a proper distance is required at position 296 for the enzyme to exhibit peptidase activity. The role of Glu-296 for the binding of bestatin, a prototype for an aminopeptidase inhibitor, was also studied. When using LTA4 as substrate, the ability of bestatin to inhibit the mutants dropped dramatically (< 0.7 % of the control), which indicates that Glu-296 is critical for binding of this inhibitor.

During catalysis, LTA4 hydrolase is suicide inactivated by its lipid substrate LTA4. The inactivation occurs via an irreversible covalent binding of LTA4 to the enzyme. Using differential peptide mapping of unmodified and suicide inactivated enzyme, a 21-residue peptide fragment (K21) encompassing the residues 365-385 of human LTA4 hydrolase, was shown to be involved in the covalent binding of LTA4 and LTA4 methyl and ethyl ester, during suicide inactivation. The degree of inactivation of both the epoxide hydrolase and the peptidase activity correlated well with the degree of peptide modification, and the competitive inhibitor bestatin could prevent enzyme inactivation and modification of K21. A modified form of peptide K21 was isolated from enzyme inactivated with LTA4 ethyl ester. Edman degradation of this peptide revealed a gap in the sequence corresponding to Tyr-378 in LTA4 hydrolase, indicating that this is the site to which LTA4 binds during suicide inactivation. Tyr-378 was exchanged to a Phe or Gln by site-directed mutagenesis.

Enzyme activity determinations of the purified mutated proteins indicated that Tyr-378 is not critical for catalyses. In fact, mutation of Tyr to Phe led to an enzyme with significantly increased turnover of LTA4. Notably, the mutated enzymes were not inactivated or covalently modified by treatment with the substrate LTA4. Moreover, the mutants were found to convert LTA4 not only to LTB4, but also into a second previously unknown enzymatic product structurally identified as 5(S),12(R) dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid, i.e. delta6-trans-delta8-cis-LTB4. Hence, Tyr-378 is a major structural determinant for suicide inactivation of LTA4 hydrolase and seems to assist in the formation of the correct double bond geometry in LTB4. Further characterization of the mutants in position 383 revealed that they exhibited a second enzymatic activity. In addition to LTB4, the mutants produced a novel metabolite with the structure 5(S),6(S)-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid.

The kinetic parameters for the formation of 5(S),6(S)-DHETE were found to be similar to those obtained for the formation of LTB4. From the stereochemical configuration of the vicinal diol it was inferred that 5(S),6(S) DHETE is formed via an SNI mechanism involving a carbocation intermediate, which in turn indicates that the enzymatic conversion of LTA4 into LTB4 follows the same mechanism. A functional and structural relationship between LTA4 hydrolase and soluble xenobiotic epoxide hydrolase (sEH)was indicated by the fact that mutants of LTA4 hydrolase and sEH convert LTA4 into 5(S),6(S)-DHETE and its epimer at C6, respectively.