title: Structure-to-function relationship of mini-lipoxygenase, a 60-kDa fragment of soybean lipoxygenase-1 with lower stability but higher enzymatic activity.
abstract: Lipoxygenase-1 (Lox-1) is a member of the lipoxygenase family, a class of dioxygenases that take part in the metabolism of polyunsatured fatty acids in eukaryotes. Tryptic digestion of soybean Lox-1 is known to produce a 60 kDa fragment, termed "mini-Lox," which shows enhanced catalytic efficiency and higher membrane-binding ability than the native enzyme (Maccarrone, M., Salucci, M. L., van Zadelhoff, G., Malatesta, F., Veldink, G. Vliegenthart, J. F. G., and Finazzi-Agrò, A. (2001) Biochemistry 40, 6819-6827). In this study, we have investigated the stability of mini-Lox in guanidinium hydrochloride and under high pressure by fluorescence and circular dichroism spectroscopy. Only a partial unfolding could be obtained at high pressure in the range 1-3000 bar at variance with guanidinium hydrochloride. However, in both cases a reversible denaturation was observed. The denaturation experiments demonstrate that mini-Lox is a rather unstable molecule, which undergoes a two-step unfolding transition at moderately low guanidinium hydrochloride concentration (0-4.5 m). Both chemical- and physical-induced denaturation suggest that mini-Lox is more hydrated than Lox-1, an observation also confirmed by 1-anilino-8-naphthalenesulfonate (ANS) binding studies. We have also investigated the occurrence of substrate-induced changes in the protein tertiary structure by dynamic fluorescence techniques. In particular, eicosatetraynoic acid, an irreversible inhibitor of lipoxygenase, has been used to mimic the effect of substrate binding. We demonstrated that mini-Lox is indeed characterized by much larger conformational changes than those occurring in the native Lox-1 upon binding of eicosatetraynoic acid. Finally, by both activity and fluorescence measurements we have found that 1-anilino-8-naphthalenesulfonate has access to the active site of mini-Lox but not to that of intact Lox-1. These findings strongly support the hypothesis that the larger hydration of mini-Lox renders this molecule more flexible, and therefore less stable.
authors.affiliation: INFM, 16152 Genova, Italy.
authors.forename: Almerinda
authors.initials: A
authors.lastname: Di Venere
authors.forename: Maria Luisa
authors.initials: ML
authors.lastname: Salucci
authors.forename: Guus
authors.initials: G
authors.lastname: van Zadelhoff
authors.forename: Gerrit
authors.initials: G
authors.lastname: Veldink
authors.forename: Giampiero
authors.initials: G
authors.lastname: Mei
authors.forename: Nicola
authors.initials: N
authors.lastname: Rosato
authors.forename: Alessandro
authors.initials: A
authors.lastname: Finazzi-Agrò
authors.forename: Mauro
authors.initials: M
authors.lastname: Maccarrone
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D001095:Arachidonic Acids / Q000737:chemistry; D001665:Binding Sites; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D008084:Lipoxygenase / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013997:Time Factors
chemical_list: D000817:Anilino Naphthalenesulfonates; D001095:Arachidonic Acids; C027132:1-anilino-8-naphthalenesulfonate; C101084:lipoxygenase L-1; D008084:Lipoxygenase; D019791:Guanidine