EVIDENCE FOR A CATALYTIC ROLE OF TYROSINE-383 IN THE PEPTIDASE REACTION OF LEUKOTRIENE-A(4) HYDROLASE

Leukotriene A(4) (LTA(4)) hydrolase is a bifunctional zinc metalloenzy me which catalyzes the find step in the biosynthesis of the proinflamm atory leukotriene B-4 and which also possesses a peptidase activity. F rom sequence comparisons with aminopeptidases, a tyrosine at position 383 in LTA(4) hydrolase has been suggested as a possible catalytic ami no acid. To explore the potential role of this amino acid in catalysis , we replaced the tyrosine residue with phenylalanine, histidine or gl utamine residues by site-directed mutagenesis. The mutated cDNAs were expressed in Escherichia coli and the resulting recombinant proteins, named [Y383F]LTA(4) hydrolase, [Y383H]LTA(4) hydrolase and [Y383Q]LTA( 4) hydrolase, were purified to homogeneity to allow assays of both the epoxide hydrolase activity, i.e. the conversion of LTA(4) into leukot riene B-4, and the peptidase activity. None of the mutated proteins ex hibited significant peptidase activities, all of them showing activiti es 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]LTA(4) hydrolase, [Y383H]LTA(4) hydrolase and [Y383Q]LTA(4) hydrolase, respectively. Kin etic analysis was performed with the mutant [Y383Q]LTA(4) hydrolase, w hich revealed an approximately 10-fold increase in K-m for leukotriene A(4) compared to that for the unmutated enzyme. At high concentration s of substrate, the difference in enzyme velocity was only moderate, w ith V-max values of 600 nmol . mg(-1) . min(-1) and 1000 nmol . mg(-1) . min(-1) for [Y383Q]LTA(4) hydrolase and the wild-type enzyme, respe ctively. No such effect of substrate concentration could be observed o n the peptidase activity. As a positive control, we exchanged a glycin e residue in position 386 for an alanine residue, and the recombinant protein, [G386A]LTA(4) 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(4) hydrolase, where it may act as a proton donor in a general base mechanism. However, our data do not allow a similar interpretatio n for the mechanism involved in the hydrolysis of LTA(4) into LTB(4).