Recombinant mouse leukotriene A4 hydrolase: a zinc metalloenzyme with dual enzymatic activities.
Recombinant mouse leukotriene A4 hydrolase was expressed in Escherichia coli as a fusion protein with ten additional amino acids at the amino terminus and was purified to apparent homogeneity by means of precipitation, anion exchange, hydrophobic interaction and chromatofocusing chromatographies. By atomic absorption spectrometry, the enzyme was shown to contain one mol of zinc/mol of enzyme. Apparent kinetic constants (Km and Vmax) for the conversion of leukotriene A4 to leukotriene B4 (at 0 degree C, pH 8) were 5 microM and 900 nmol/mg per min, respectively. The purified enzyme also exhibited significant peptidase activity towards the synthetic amide alanine-4-nitroanilide. Km and Vmax for this reaction (at 37 degrees C, pH 8) were 680 microM and 365 nmol/mg per min, respectively. Apo-leukotriene A4 hydrolase, prepared by treating the enzyme with 1,10-phenanthroline, was virtually inactive with respect to both enzymatic activities, but could be reactivated by addition of stoichiometric amounts of zinc or cobalt. Exposure of the enzyme to leukotriene A4 resulted in a dose-dependent inactivation of both enzyme activities.
Department of Physiological Chemistry, Karolinska Institutet, Stockholm, Sweden.
A
A
Wetterholm
J F
JF
Medina
O
O
Rådmark
R
R
Shapiro
J Z
JZ
Haeggström
B L
BL
Vallee
B
B
Samuelsson
D000818:Animals; D001051:Apoenzymes / Q000302:isolation & purification / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004851:Epoxide Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013054:Spectrophotometry, Atomic; D015032:Zinc / Q000032:analysis*
D001051:Apoenzymes; D011994:Recombinant Proteins; D004851:Epoxide Hydrolases; D015032:Zinc; C043134:leukotriene A4 hydrolase