Leukotriene A4 hydrolase, mutation of tyrosine 378 allows conversion of leukotriene A4 into an isomer of leukotriene B4.
Leukotriene A4 hydrolase catalyzes the final step in the biosynthesis of the proinflammatory compound leukotriene B4, a reaction which is accompanied by suicide inactivation of the enzyme by leukotriene A4. We have recently reported that Tyr-378 is a major structural determinant for suicide inactivation and that mutation of Tyr-378 into Phe or Gln protects leukotriene A4 hydrolase from this catalytic restriction (Mueller, M. J., Blomster, M., Opperman, U. C. T., Jörnvall, H., Samuelsson, B., and Haeggström, 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 A4 hydrolase converts leukotriene A4 not only into leukotriene B4 but also into a second, previously unknown, product of the enzyme. From biophysical analyses and comparison with a synthetic standard, the structure of this product was determined to 5S,12R-dihydroxy-6,10-trans-8, 14-cis-eicosatetraenoic acid, i.e. Delta6-trans-Delta8-cis-leukotriene B4. The relative formation of Delta6-trans-Delta8-cis-leukotriene B4 versus leukotriene B4 by [Y378F]- and [Y378Q]leukotriene A4 hydrolase, was 18% and 32%, respectively. For [Y378F]leukotriene A4 hydrolase, the turnover of leukotriene A4 into leukotriene B4 or Delta6-trans-Delta8-cis-leukotriene B4 was calculated to 2.5 s-1 which is almost three times the kcat value of the wild type enzyme. Taken together, these findings indicate that Tyr-378 is located at the active site where it assists in the formation of the correct double-bond geometry in the product leukotriene B4.
Mueller
M J
MJ
Department of Medical Biochemistry and Biophysics, Division of Chemistry II, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Andberg
M B
MB
Samuelsson
B
B
Haeggström
J Z
JZ
D001665:Binding Sites; D002384:Catalysis; D004851:Epoxide Hydrolases / Q000235:genetics / Q000378:metabolism*; D007536:Isomerism; D017572:Leukotriene A4 / Q000378:metabolism*; D007975:Leukotriene B4 / Q000096:biosynthesis*; D016297:Mutagenesis, Site-Directed; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
D017572:Leukotriene A4; D007975:Leukotriene B4; D014443:Tyrosine; D004851:Epoxide Hydrolases; C043134:leukotriene A4 hydrolase