Open AccessEvidence for a Catalytic Role of Tyrosine 383 in the Peptidase Reaction of Leukotriene A 4 Hydrolase
Author(s) -
Blomster Martina,
Wetterholm Anders,
Mueller Martin J.,
Haeggström Jesper Z.
Publication year - 1995
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1995.0528d.x
Subject(s) - epoxide hydrolase , hydrolase , epoxide hydrolase 2 , chemistry , biochemistry , enzyme , phosphofructokinase 2 , site directed mutagenesis , tyrosine , stereochemistry , active site , histidine , mutant , gene , microsome
Leukotriene A 4 (LTA 4 ) hydrolase is a bifunctional zinc metalloenzyme which catalyzes the final step in the biosynthesis of the proinflammatory leukotriene B 4 and which also possesses a peptidase activity. From sequence comparisons with aminopeptidases, a tyrosine at position 383 in LTA 4 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 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 leukotriene B 4 , 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]LTA 4 hydrolase, [Y383H]LTA 4 hydrolase and [Y383Q]LTA 4 hydrolase, respectively. Kinetic analysis was performed with the mutant [Y383Q]LTA 4 hydrolase, which revealed an approximately 10‐fold increase in K m for leukotriene A 4 compared to that for the unmutated enzyme. At high concentrations of substrate, the difference in enzyme velocity was only moderate, with 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, 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]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 interpretation for the mechanism involved in the hydrolysis of LTA 4 into LTB 4 .