LEUKOTRIENE A(4) HYDROLASE, MUTATION OF TYROSINE-378 ALLOWS CONVERSION OF LEUKOTRIENE A(4) INTO AN ISOMER OF LEUKOTRIENE B-4

Leukotriene A(4) hydrolase catalyzes the final step in the biosynthesi s of the proinflammatory compound leukotriene B-4, a reaction which is accompanied by suicide inactivation of the enzyme by leukotriene A(4) . We have recently reported that Tyr-378 is a major structural determi nant for suicide inactivation and that mutation of Tyr-378 into Phe or Gln protects leukotriene A(4) hydrolase from this catalytic restricti on (Mueller, M. J., Blomster, M., Opperman, U. C. T., Jornvall, H., Sa muelsson, B., and Haeggstrom, J. Z. (1996) Proc. Natl. Acad. Sci. U. S . A. 93, 5931-5935), In the present study, we show that both [Y378F]- and [Y378Q]leukotriene A(4) hydrolase converts leukotriene A(4) not on ly into leukotriene B-4 but also into a second, previously unknown, pr oduct of the enzyme. From biophysical analyses and comparison with a s ynthetic standard, the structure of this product was determined to 12R -dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid, i.e. Delta(6)-tr ans-Delta(8)-cis-leukotriene B-4. The relative formation of Delta(6)-t rans-Delta(8)-cis-leukotriene B-4 versus leukotriene B-4 by [Y378F]- a nd [Y378Q]leukotriene A(4) hydrolase, was 18% and 32%, respectively. F or [Y378F]leukotriene A(4) hydrolase, the turnover of leukotriene A(4) into leukotriene B-4 or Delta(6)-trans-Delta(8)-cis-leukotriene B-4 w as calculated to 2.5 s(-1) which is almost three times the k(cat) valu e of the wild type enzyme, Taken together, these findings indicate tha t Tyr-378 is located at the active site where it assists in the format ion of the correct double-bond geometry in the product leukotriene B-4 .