Leukotriene A4 hydrolase: protection from mechanism-based inactivation by mutation of tyrosine-378.

Leukotriene A4 (LTA4) hydrolase [(7E,9E,11Z,14Z)-(5S,6S)-5,6-epoxyicosa-7, 9,11,14-tetraenoate hydrolase; EC 3.3.2.6] is a bifunctional zinc metalloenzyme that catalyzes the final step in the biosynthesis of the potent chemotactic agent leukotriene B4 (LTB4). LTA4 hydrolase/aminopeptidase is suicide inactivated during catalysis via an apparently mechanism-based irreversible binding of LTA4 to the protein in a 1:1 stoichiometry. Previously, we have identified a henicosapeptide, encompassing residues Leu-365 to Lys-385 in human LTA4 hydrolase, which contains a site involved in the covalent binding of LTA4 to the native enzyme. To investigate the role of Tyr-378, a potential candidate for this binding site, we exchanged Tyr for Phe or Gln in two separate mutants. In addition, each of two adjacent and potentially reactive residues, Ser-379 and Ser-380, were exchanged for Ala. The mutated enzymes were expressed as (His)6-tagged fusion proteins in Escherichia coli, purified to apparent homogeneity, and characterized. Enzyme activity determinations and differential peptide mapping, before and after repeated exposure to LTA4, revealed that wild-type enzyme and the mutants [S379A] and [S380A]LTA4hydrolase were equally susceptible to suicide inactivation whereas the mutants in position 378 were no longer inactivated or covalently modified by LTA4. Furthermore, in [Y378F]LTA4 hydrolase, the value of kcat for epoxide hydrolysis was increased 2.5-fold over that of the wild-type enzyme. Thus, by a single-point mutation in LTA4 hydrolase, catalysis and covalent modification/inactivation have been dissociated, yielding an enzyme with increased turnover and resistance to mechanism-based inactivation.

Leukotriene A4 hydrolase: mapping of a henicosapeptide involved in mechanism-based inactivation.

Leukotriene A4 (LTA4) hydrolase [7E,9E,11Z,14Z)-(5S,6S)-5,6-epoxyicosa-7,9 ,11,14-tetraenoate hydrolase; EC 3.3.2.6] is a bifunctional zinc metalloenzyme which converts LTA4 into the chemotactic agent leukotriene B4 (LTB4). Suicide inactivation, a typical feature of LTA4 hydrolase/aminopeptidase, occurs via an irreversible, apparently mechanism-based, covalent binding of LTA4 to the protein in a 1:1 stoichiometry. Differential lysine-specific peptide mapping of unmodified and suicide-inactivated LTA4 hydrolase has been used to identify a henicosapeptide, encompassing the amino acid residues 365-385 of human LTA4 hydrolase, which is involved in the binding of LTA4, LTA4 methyl ester, and LTA4 ethyl ester to the native enzyme. A modified form of this peptide, generated by lysine-specific digestion of LTA4 hydrolase inactivated by LTA4 ethyl ester, could be isolated for complete Edman degradation. The sequence analysis revealed a gap at position 14, which shows that binding of the leukotriene epoxide had occurred via Tyr-378 in LTA4 hydrolase. Inactivation of the epoxide hydrolase and the aminopeptidase activity was accompanied by a proportionate modification of the peptide. Furthermore, both enzyme inactivation and peptide modification could be prevented by preincubation of LTA4 hydrolase with the competitive inhibitor bestatin, which demonstrates that the henicosapeptide contains functional elements of the active site(s). It may now be possible to clarify the molecular mechanisms underlying suicide inactivation and epoxide hydrolysis by site-directed mutagenesis combined with structural analysis of the lipid molecule, covalently bound to the peptide.

Evidence for a catalytic role of tyrosine 383 in the peptidase reaction of leukotriene A4 hydrolase.

Leukotriene A4 (LTA4) hydrolase is a bifunctional zinc metalloenzyme which catalyzes the final step in the biosynthesis of the proinflammatory leukotriene B4 and which also possesses a peptidase activity. From sequence comparisons with aminopeptidases, a tyrosine at position 383 in LTA4 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]LTA4 hydrolase, [Y383H]LTA4 hydrolase and [Y383Q]LTA4 hydrolase, were purified to homogeneity to allow assays of both the epoxide hydrolase activity, i.e. the conversion of LTA4 into leukotriene B4, 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]LTA4 hydrolase, [Y383H]LTA4 hydrolase and [Y383Q]LTA4 hydrolase, respectively. Kinetic analysis was performed with the mutant [Y383Q]LTA4 hydrolase, which revealed an approximately 10-fold increase in Km for leukotriene A4 compared to that for the unmutated enzyme. At high concentrations of substrate, the difference in enzyme velocity was only moderate, with Vmax values of 600 nmol.mg-1.min-1 and 1000 nmol.mg-1.min-1 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 LTA4 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 LTA4 into LTB4.

Synthesis and processing of the rubella virus p110 polyprotein precursor in baculovirus-infected Spodoptera frugiperda cells.

In order to study the processing of rubella virus (RV) structural proteins (capsid protein, of 33 kDa; E2 of 42-47 kDa; and E1 of 58 kDa) in Spodoptera frugiperda (fall armyworm) cells, a 24S cDNA encoding the polyprotein precursor, p110, was inserted under the transcriptional regulation of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed during viral infection. By immunoblot analysis using antibodies directed against whole RV and the individual structural proteins, evidence is presented that polypeptides similar to those synthesized in RV-infected B-Vero cells are expressed in this lepidopteran insect cell line infected with the recombinant baculovirus, VL1392-RV24S. The identity of the recombinant proteins was further confirmed using human convalescent sera. By expressing the recombinant proteins in the presence and absence of tunicamycin, we have further demonstrated that the 24S transcription-translation unit of RV, is expressed and proteolytically cleaved similarly, if not identically, in Sf9 cells as compared to B-Vero cells.