Site-Directed Mutagenesis of Lysine 125 in Malate Dehydrogenase

Malate dehydrogenase is a multimeric enzyme among living organisms that catalyzes the reverse transformation of malate and oxaloacetate using the reduction of NAD+ to NADH. This reaction plays a role in metabolic pathways including the citric acid cycle, gluconeogenesis, and anaerobic metabolism. MDH shares a similar 3-dimensional structure and mechanism with lactate dehydrogenase. Knowing the structure is important when it comes to the redesign of enzyme mutations, which can be a useful method for studying the catalysis of small substrates. Physiological effects of the amino acid sequence alterations are easier to predict when the structure is known. The active site of MDH consists of a hydrophobic vacuole containing binding site for the substrate and nicotinamide ring of the coenzyme. Within the active site there is a loop region containing amino acids 119-137. The active site exhibits an open conformation when the substrate or cofactor is bound and a closed conformation when nothing is bound. The charges within the loop region position the substrate in the correct orientation for efficient catalysis. It was shown that Lysine125, within the loop region of MDH, made essential interactions with co-factor and nearby residues that may have been involved in catalysis (Shania, 2019). Shown in figure 1, Lys125 and R124 are in close proximity with each other. Since both molecules have a positive charge, they are repelling against each other. We are predicting that the position of Lys125 and R124 are causing G263 to have a less stable hydrogen bond. We hypothesized that if Alanine replaces Lysine at position 125, then Arg124 will have a better position and be more stably bound to G263 resulting in a better guide for the substrate to the active site.https://digitalcommons.morris.umn.edu/urs_2023/1003/thumbnail.jp

Nanoemulsion stability: experimental evaluation of the flocculation rate from turbidity measurements

The coalescence of liquid drops induces a higher level of complexity compared to the classical studies about the aggregation of solid spheres. Yet, it is commonly believed that most findings on solid dispersions are directly applicable to liquid mixtures. Here, the state of the art in the evaluation of the flocculation rate of these two systems is reviewed. Special emphasis is made on the differences between suspensions and emulsions. In the case of suspensions, the stability ratio is commonly evaluated from the initial slope of the absorbance as a function of time under diffusive and reactive conditions. Puertas and de las Nieves (1997) developed a theoretical approach that allows the determination of the flocculation rate from the variation of the turbidity of a sample as a function of time. Here, suitable modifications of the experimental procedure and the referred theoretical approach are implemented in order to calculate the values of the stability ratio and the flocculation rate corresponding to a dodecane-in-water nanoemulsion stabilized with sodium dodecyl sulfate. Four analytical expressions of the turbidity are tested, basically differing in the optical cross section of the aggregates formed. The first two models consider the processes of: a) aggregation (as described by Smoluchowski) and b) the instantaneous coalescence upon flocculation. The other two models account for the simultaneous occurrence of flocculation and coalescence. The latter reproduce the temporal variation of the turbidity in all cases studied (380 \leq [NaCl] \leq 600 mM), providing a method of appraisal of the flocculation rate in nanoemulsions