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,.

Enzymes and receptors in the leukotriene cascade

“…Thus, mutagenetic replacements of Glu-296 in LTA 4 hydrolase, a conserved residue within the zinc signature, abrogated only the peptidase activity and not the ability to catalyze the conversion of LTA 4 into LTB 4 , which suggests a direct catalytic role for Glu-296 in the peptidase reaction, possibly as a general base [76,77]. Furthermore, sequence comparisons and mutational analysis have demonstrated that Tyr-383 plays an important role in the peptidase reaction of LTA 4 hydrolase, presumably as a proton donor [78]. Further investigation of the catalytic properties of mutants in position 383 revealed the formation of large quantities of a novel metabolite of LTA 4 structurally identified as 5S,6S-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid (5S,6S-DHETE), in addition to the expected LTB 4 [79].…”

Evolutionary aspects of lipoxygenases and genetic diversity of human leukotriene signaling

“…Three amino acids (His295, His299 and Glu318) form the Znbinding cluster and mutagenesis studies indicated that the loss of one of these residues blocks the catalytic activity [323][324][325][326]. LTA4H undergoes suicidal inactivation during LTA4 hydrolysis and as molecular reason for the loss in catalytic activity chemical modification of Tyr378 was suggested [327,328]. Additional mechanistic studies indicated the importance of the following amino acids for the functionality of the enzyme: Gln136, Glu271, Glu296, Asp375, Arg563, and Lys565 [6,[329][330][331].…”

Crystal structures of leukotriene A4 hydrolase in complex with captopril and two competitive tight‐binding inhibitors

“…Human recombinant LTA4H was expressed in Escherichia coli and purified to near homogeneity, as previously described (13,30). Greater purity (>95%) was achieved by chromatography on hydroxyapatite (TSKgel HA-1000, Tosoh Biosep GmbH, Stuttgart, Germany) and anion-exchange (Mono-Q HR5/5) resins.…”