Evidence for a Catalytic Role of Tyrosine 383 in the Peptidase Reaction of Leukotriene A₄ Hydrolase

Abstract

Leukotriene A, (LTAJ hydrolase is a bifunctional zinc metalloenzyme which catalyzes the final step in the biosynthesis of the proinflammatory leukotriene B, and which also possesses a peptidase activity. From sequence comparisons with aminopeptidases, a tyrosine at position 383 in LTA, hydrolase has been suggested as a possible catalytic amino acid. To explore the potential role of this amino acid in catalysis, we replaced the tyrosine residue with phenylalanine, histidine or glutamine residues by site-directed mutagenesis. The mutated cDNAs were expressed in Escherichiu coli and the resulting recombinant proteins, named [Y383F]LTA, hydrolase, [Y383H]LTA, hydrolase and [Y383Q]LTA4 hydrolase, were purified to homogeneity to allow assays of both the epoxide hydrolase activity, i.e. the conversion of LTA, into leukotriene B,, and the peptidase activity.None of the mutated proteins exhibited significant peptidase activities, all of them showing activities less than 0.3 % that of the wild-type enzyme. The epoxide hydrolase activity was not affected to the same degree and corresponded to 11, 16 and 17% that of the unmutated enzyme for [Y383F]LT& hydrolase, [Y383H]LTA, hydrolase and [Y383Q]LTA4 hydrolase, respectively. Kinetic analysis was performed with the mutant [Y383Q]LTA4 hydrolase, which revealed an approximately 10-fold increase in K,,, for leukotriene A, compared to that for the unmutated enzyme. At high concentrations of substrate, the difference in enzyme velocity was only moderate, with V,,, values of 600 nmol . mg-' . min-' and 1000 nmol . mg-I . min-' for [Y383Q]LTA4 hydrolase and the wild-type enzyme, respectively. No such effect of substrate concentration could be observed on the peptidase activity. As a positive control, we exchanged a glycine residue in position 386 for an alanine residue, and the recombinant protein, [G386A]LTA4 hydrolase retained 19% and 77% of the peptidase and epoxide hydrolase activities, respectively. The results from this study are consistent with a role for Tyr383 in the peptidase reaction of LTA, hydrolase, where it may act as a proton donor in a general base mechanism. However, our data do not allow a similar interpretation for the mechanism involved in the hydrolysis of LTA, into LTB,.

Aminopeptidase B from the rat testis is a bifunctional enzyme structurally related to leukotriene-A4 hydrolase

“…In LTA 4 hydrolase, two tyrosine residues (Y 379 and Y 384 ) were implicated in the suicide inactivation of the enzyme and in catalysis, respectively. Indeed, the first one covalently binds the LTA 4 substrate to inactivate the enzyme (28), and the latter is necessary to the peptidase activity as a proton donor in a general base mechanism (34). The conservation of these two residues in Ap-B further supports its in vitro, and probably in vivo, bifunctionality.…”

Epoxide hydrolases: biochemistry and molecular biology

“…However, the epoxide hydrolase function was not impacted to the same degree, corresponding to 11,16 4 hydrolase, where it may act as a proton donor in a general base mechanism. These and other data provide evidence that the two catalytic activities of leukotriene A 4 hydrolase involve non-identical but overlapping active sites [29,30]. Evidence for a carbocation intermediate has been reported in the enzymatic transformation of leukotriene A 4 into leukotriene B 4 [31].…”

“…From sequence comparisons with aminopeptidases, a tyrosine at position 383 in leukotriene A 4 hydrolase identified as a possible catalytic residue and tested in this respect using site-specific mutagenesis to substitute this position with phenylalanine, histidine or glutamine [29]. The mutated proteins exhibited peptidase activities B 0.3% that of the wild-type enzyme.…”

Molecular Cloning and Functional Expression of aCaenorhabditis elegans Aminopeptidase Structurally Related to Mammalian Leukotriene A4 Hydrolases

“…Members of this family also include aminopeptidase A, aminopeptidase N, cysteine aminopeptidase, and LTA 4 hydrolase. The tyrosine residue Tyr 383 in both the human and the mouse sequences, which is essential for the peptidase activity of the mammalian LTA 4 hydrolase/aminopeptidase enzyme and may act as a proton donor in a general base mechanism (37), is conserved in the C. elegans AP-1 (Fig. 2, Tyr 387 in the C. elegans sequence).…”