Characterization of the interdependency between residues that bind the substrate in a β-glycosidase

The manner by which effects of simultaneous mutations combine to change enzymatic activity is not easily predictable because these effects are not always additive in a linear manner. Hence, the characterization of the effects of simultaneous mutations of amino acid residues that bind the substrate can make a significant contribution to the understanding of the substrate specificity of enzymes. In the β-glycosidase from Spodoptera frugiperda (Sfβgly), both residues Q39 and E451 interact with the substrate and this is essential for defining substrate specificity. Double mutants of Sfβgly (A451E39, S451E39 and S451N39) were prepared by site-directed mutagenesis, expressed in bacteria and purified using affinity chromatography. These enzymes were characterized using p-nitrophenyl β-galactoside and p-nitrophenyl β-fucoside as substrates. The k cat/Km ratio for single and double mutants of Sfβgly containing site-directed mutations at positions Q39 and E451 was used to demonstrate that the effect on the free energy of ES‡ (enzyme-transition state complex) of the double mutations (∆∆G‡xy) is not the sum of the effects resulting from the single mutations (∆∆G‡x and ∆∆G‡y). This difference in ∆∆G‡ indicates that the effects of the single mutations partially overlap. Hence, this common effect counts only once in ∆∆G‡xy. Crystallographic data on β-glycosidases reveal the presence of a bidentate hydrogen bond involving residues Q39 and E451 and the same hydroxyl group of the substrate. Therefore, both thermodynamic and crystallographic data suggest that residues Q39 and E451 exert a mutual influence on their respective interactions with the substrate.

Outsourcing decision-making aspects considered by IT departments in Brazilian companies

The study of Information Technology (IT) outsourcing is relevant because companies are outsourcing their activities more than ever. An important IT outsourcing research area is the decision-making process. In other words, the comprehension of how companies decide about outsourcing their IT operations is relevant from research point of view. Therefore, the objective of this study is to understand the decision-making process used by Brazilian companies when outsourcing their IT operations. An analysis of the literature that refers to this subject showed that six aspects are usually considered by companies on the evaluation of IT outsourcing service alternatives. This research verified how these six aspects are considered by Brazilian companies on IT outsourcing decisions. The survey showed that Brazilian companies consider all the six aspects, but each of them has a different level of importance. The research also grouped the aspects according to their level of importance and interdependency, using factorial analysis to understand the logic behind IT outsourcing decision process. (C) 2009 Elsevier B.V. All rights reserved.