Properties As Enzyme A Specific Mechanism of Action

As shown by Figure 2, the mechanism of action is an iron-catalyzed one-electron redox cycle that causes free radical oxidation of polyunsaturated fatty acids. X-ray crystallography revealed many clues of the details of catalysis. Not obvious in the structural depiction of the LOX protein shown in Figure 3 is a conical hydropho-bic cavity, or tunnel, that connects the bottom of the enzyme with the iron active site, which is believed to be a path for O2. A substrate channel was identified by a long, narrow hydrophobic cavity with two bends lying to the right of the iron (75). As discussed above, the iron active site serves to cycle electrons. Iron is com-plexed by three histidines and one isoleucine. Higherresolution x-ray crystallographic analysis showed that iron was additionally liganded with one water and weakly with asparagine694 (76), making a total of six coordinates (Fig. 4). It has been proposed that the

Figure 3 The three-dimensional structure of soybean lipox-ygenase-1 determined by x-ray crystallography. The iron active site is shown as a sphere, a-helices by cylinders, strands in ^-sheets by arrows, coils by narrow rods. (From Ref. 75.)

Figure 4 The six-coordinate ligands of the iron active site of soybean LOX-1 showing five amino acids with their numbered positions in the sequence; the sixth ligand (H2O) is thought to abstract hydrogen from substrate (OH- to H2O transition) as shown. The position of the three histidines and isoleucine was determined by x-ray crystallography (67), and high-resolution x-ray analysis showed the presence of H2O and asparagine as ligands (76). The OH- to H2O transition has been proposed by a number of investigators (e.g., see 15).

Figure 4 The six-coordinate ligands of the iron active site of soybean LOX-1 showing five amino acids with their numbered positions in the sequence; the sixth ligand (H2O) is thought to abstract hydrogen from substrate (OH- to H2O transition) as shown. The position of the three histidines and isoleucine was determined by x-ray crystallography (67), and high-resolution x-ray analysis showed the presence of H2O and asparagine as ligands (76). The OH- to H2O transition has been proposed by a number of investigators (e.g., see 15).

water ligand exists as a hydroxyl anion/water transition that accepts hydrogen from the substrate (see review: 15). Asparagine694 (or its equivalent in sequential position) is mostly conserved in various LOXs, but not completely (15); replacement by histidine, as observed in certain LOXs of mammalian origin, reduced kcat significantly (77).

B. Kinetics, Km, kcat, Activation Energy

A number of factors must be considered prior to determining LOX kinetics. Kinetics should be determined below the critical micelle concentration (CMC), which is dependent principally on the pH, and the presence of other hydrophobic substances, including surfactants. The CMC for linoleic acid has been reported for Na borate buffer (0.1 M) at 167, 60, and 21 ^M at pH 10, 9, and 8, respectively (78). Ca2+ has been reported as a cofactor of LOX, but this ion is ineffective below the CMC, indicating that it affects CMC by calcium salt formation (79). However, Ca2+ may not be completely without effect in vivo. It has been recently reported that Ca2+ stimulated the membrane binding of soybean LOX-1, implying a regulatory mechanism for Ca2+ (80).

An initial lag in the kinetics of LOX must also be considered. The lag can be overcome simply by adding a small quantity of product hydroperoxide (81). This can be interpreted as an initial oxidation of native Fe2+-LOX by hydroperoxide product to give Fe3+-LOX to prime the catalytic cycle, but detailed kinetic analysis suggested a somewhat more complex interpretation (23).

The turnover rate, kcat, and Km for soybean LOX-1 with linoleic acid substrate has been variously reported at 280-350 sec-1 and 12-25 ^M, respectively (15, 81, and Refs. therein).

The activation energy (Ea) for soybean LOX-1 and linoleic acid was determined to be 22 kcal/mol, and AH= = 21 kcal/mol, AS= = 20 e.u., and AG= ' 15 kcal/mol (82, 83).

A kinetic scheme has been developed (23 and Refs. therein) and was recently modified by Solomon et al. (15) to account for recent findings of the active site. Figure 2 essentially illustrates this scheme modified to clearly show the aerobic and O2-starved aspects of the active site redox cycle, as well as the products obtained.

C. Effect of pH, Inhibitors, and Surfactants

Soybean LOX-1 is generally stable over a wide range of pHs. Its activity is optimum between pH 9 and 10.

Activity is negligible at pH 6.5, but it is stable at pHs as low as 4.5. Adjustment to pH 4.5 has been used with crude preparations to remove unwanted protein in LOX purification (84). However, pH 3.0 and below causes irreversible inactivation of soybean LOX activity (85).

Although many inhibitors of LOX have been investigated, only representative examples can be cited here. First, it should be emphasized that soybean LOX catalyzes its own destruction during oxidation of substrate. The destruction is greater with substrates of a higher degree of unsaturation (86). Several types of inhibitors mainly can be categorized as follows (for citations also see 86 and Refs. therein): (a) substrate suicide inhibitors, such as acetylenic fatty acids (irreversible) (87); (b) chain-breaking antioxidants (usually competitive and reversible) (88); (c) iron chelators (often reversible) (89); (d) disrupters of the active site (90); (e) reductants of the active-site iron (91); (f) free radical reactions with LOX, such as with hydroxyl radicals produced from H2O2 (presumably irreversible)

(92); and (g) substrate mimics (usually competitive)

(93). In regard to substrate mimics, such as various fatty acids, potential errors are possible because of their disruption of CMC stability (94). That is, some fatty acids actually may not be inhibitors, and exert their effect by changing the CMC of the substrate.

Nonionic surfactants, such as Tween 20 and Triton X-100, are often used by researchers to clarify substrate solutions. However, it has been found that surfactants actually decrease activity, except when concentrations of substrate and surfactant are high and low, respectively (95). Kinetic data suggested that surfactant sequestered substrate in micelles, whereas the actual substrate was indicated to be sol-vated monomers. That is, substrate could be oxidized only after escaping micelles in equilibrium with the aqueous phase. The rate increase at high substrate and low surfactant concentrations was interpreted as alleviating substrate inhibition by sequestering substrate in micelles. Besides surfactants, ethanol or methanol is often used to conveniently disperse fatty acid substrates, but alcohols also inhibited the activity of LOX, increased the Km and decreased the Vmax (96). The alcohol effect was found to be reversible.

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