Maja Pavela‐Vrančič

In plant tissues, enzymes implicated in the lipoxygenase (LOX) pathway are responsible for the hydroperoxydation of polyunsaturated fatty acids, ultimately leading to the production of small chemical species involved in several physiological processes. During industrial olive oil production, these enzymes are activated upon crushing and grinding of olive fruit tissue, subsequently leading to the synthesis of volatile compounds responsible for the positive aroma and flavor of the oil. An investigation of LOX activity during olive fruit ripening and malaxation could assist in the production of oils with favorable aroma and taste. Therefore, a reliable method for olive LOX purification is crucial. Here we report a critical review of six LOX extraction protocols, two of which have shown minimum enzyme activity, possibly leading to misconceptions in the interpretation of experimental data. Future research concerning olive LOX should employ extraction methods that preserve enzyme activity.

The adenylation (A) domain in nonribosomal peptide synthetases catalyses a two‐step reaction in which an amino acid is activated and then transferred to the neighbouring thiolation (T) domain. In this study, we investigated the role of the conserved A9 core sequence of the A‐domain of tyrocidine synthetase 1, by analysis of single amino acid mutations in the A9 region. Mutation of an absolutely conserved proline (P490G) significantly reduced the conformational stability of the protein, as evidenced by increased susceptibility to proteolytic cleavage and denaturation. All mutant A‐domains were capable of amino acid activation, but the activity in the overall reaction was reduced. Surprisingly, the S491R mutant (mutation at the first residue following the A9 motif) showed elevated overall activity compared to the wild‐type protein. Our results suggest that the A9 core sequence plays a role in the second reaction step, in which it could serve as a “clip” for the proper positioning of residues important for the interaction with the T‐domain, and/or stabilisation of the thioester‐forming conformation.

Although alkaline phosphatase (APase) from Escherichia coli crystallizes as a symmetric dimer, it displays deviations from Michaelis–Menten kinetics, supported by a model describing a dimeric enzyme with unequal subunits [Orhanović S., Pavela‐Vrančič M. and Flogel‐Mršić M. (1994) Acta. Pharm.44, 87–95]. The possibility, that the observed asymmetry could be attributed to negative cooperativity in Mg²⁺ binding, has been examined. The influence of the metal ion content on the catalytic properties of APase from E. coli has been examined by kinetic analyses. An activation study has indicated that Mg²⁺ enhances APase activity by a mechanism that involves interactions between subunits. The observed deviations from Michaelis–Menten kinetics are independent of saturation with Zn²⁺ or Mg²⁺ ions, suggesting that asymmetry is an intrinsic property of the dimeric enzyme. In accordance with the experimental data, a model describing the mechanism of substrate hydrolysis by APase has been proposed. The release of the product is enhanced by a conformational change generating a subunit with lower affinity for both the substrate and the product. In the course of the catalytic cycle the conformation of the subunits alternates between two states in order to enable substrate binding and product release. APase displays higher activity in the presence of Mg²⁺, as binding of Mg²⁺ increases the rate of conformational change. A conformationally controlled and Mg²⁺‐assisted dissociation of the reaction product (P_i) could serve as a kinetic switch preventing loss of P_i into the environment.