Kinetic investigations of the rate-limiting step in human 12- and 15-lipoxygenase.
Mammalian lipoxygenases have been implicated in several inflammatory disorders; however, the details of the kinetic mechanism are still not well understood. In this paper, human platelet 12-lipoxygenase (12-hLO) and human reticulocyte 15-lipoxygenase-1 (15-hLO) were tested with arachidonic acid (AA) and linoleic acid (LA), respectively, under a variety of changing experimental conditions, such as temperature, dissolved oxygen concentration, and viscosity. The data that are presented show that 12-hLO and 15-hLO have slower rates of product release (k(cat)) than soybean lipoxygenase-1 (sLO-1), but similar or better rates of substrate capture for the fatty acid (k(cat)/K(M)) or molecular oxygen [k(cat)/K(M(O)2)]. The primary, kinetic isotope effect (KIE) for 15-hLO with LA was determined to be temperature-independent and large ((D)k(cat) = 40 +/- 8), over the range of 10-35 degrees C, indicating that C-H bond cleavage is the sole rate-limiting step and proceeds through a tunneling mechanism. The (D)k(cat)/K(M) for 15-hLO, however, was temperature-dependent, consistent with our previous results [Lewis, E. R., Johansen, E., and Holman, T. R. (1999) J. Am. Chem. Soc. 121, 1395-1396], indicating multiple rate-limiting steps. This was confirmed by a temperature-dependent, k(cat)/K(M) solvent isotope effect (SIE), which indicated a hydrogen bond rearrangement step at low temperatures, similar to that of sLO-1 [Glickman, M. H., and Klinman, J. P. (1995) Biochemistry 34, 14077-14092]. The KIE could not be determined for 12-hLO due to its inability to efficiently catalyze LA, but the k(cat)/K(M) SIE was temperature-independent, indicating distinct rate-limiting steps from both 15-hLO and sLO-1.
Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.
Erika N
EN
Segraves
Theodore R
TR
Holman
D001092:Arachidonate 12-Lipoxygenase / Q000737:chemistry* / Q000378:metabolism; D001093:Arachidonate 15-Lipoxygenase / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D005227:Fatty Acids / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008041:Linoleic Acids / Q000378:metabolism; D008084:Lipoxygenase / Q000737:chemistry / Q000378:metabolism; D010920:Placenta / Q000201:enzymology*; D012156:Reticulocytes / Q000201:enzymology*; D013025:Glycine max / Q000201:enzymology; D013379:Substrate Specificity; D013696:Temperature; D014783:Viscosity
D005227:Fatty Acids; D008041:Linoleic Acids; D008084:Lipoxygenase; D001092:Arachidonate 12-Lipoxygenase; D001093:Arachidonate 15-Lipoxygenase