Chemical modification of leukotriene A4 hydrolase. Indications for essential tyrosyl and arginyl residues at the active site.
We have employed chemical modification to identify amino acids essential for the catalytic activities of the bifunctional zinc metalloenzyme leukotriene A4 hydrolase (EC 3.3.2.6). The epoxide hydrolase and the peptidase activity were both rapidly inactivated by N-acetylimidazole and tetranitromethane. Furthermore, treatment with 2,3-butanedione and phenylglyoxal also resulted in loss of both activities. Leukotriene A4 hydrolase could be protected from inactivation by these tyrosyl and arginyl reagents by the competitive inhibitors bestatin and captopril, respectively. Two tyrosyl and three arginyl residues were found by differential labeling techniques to be protected by the inhibitors, which thus suggested that these amino acids are located close to or at the active center of the enzyme. Limited modification by thiol reagents and particularly methyl methanethiosulfonate led to a > 10-fold increase in the peptidase activity and a decreased epoxide hydrolase activity, whereas prolonged treatment inhibited both activities. Kinetic analysis of modified enzyme, using the substrate alanine p-nitroanilide, revealed that the stimulatory effect on the peptidase activity was due to increased enzyme displayed a reduced apparent affinity constant for chloride ions, which strongly stimulate the peptidase activity. Neither activation nor inactivation by methyl methanethiosulfonate was influenced by the presence of competitive inhibitors, which suggested that this compound did not react with amino acids at the active center but rather with residues of importance for the overall enzyme conformation.
Mueller
M J
MJ
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Samuelsson
B
B
Haeggström
J Z
JZ
D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites; D003931:Diacetyl / Q000494:pharmacology; D004851:Epoxide Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D007093:Imidazoles / Q000494:pharmacology; D010658:Phenylglyoxal / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013774:Tetranitromethane / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*
D007093:Imidazoles; D013439:Sulfhydryl Reagents; D014443:Tyrosine; D001120:Arginine; D004851:Epoxide Hydrolases; D013774:Tetranitromethane; D003931:Diacetyl; D010658:Phenylglyoxal; C043134:leukotriene A4 hydrolase; C012160:N-acetylimidazole