Isolation and Characterization of Leishmanial Adenine Aminohydrolase as a Drug Target.

Background: Search for new drug targets is becoming imperative these days, given that marketed chemotherapeutic drugs have lost their efficacy against harmful agents because of adaptability to climatic changes and co-evolving vectors to new hosts. In the wake of such a challenge, the prominence of biochemical studies is increasing by way of exploring selective enzymes and investigating their structural and functional properties through biochemical kinetic parameter Km for the application of IC50 using designed drugs. Recently, discovered Adenine Aminohydrolase (EC 3.5.4.2) in Leishmania has been found to be absent in mammalian purine salvage pathway and thus considered as a promising drug target against infectious agents.
Objectives: The objective of this study is to isolate and characterize AAH by learning its kinetic mode of action using preferred substrate Adenine and additives estimated through expected product formation Hypoxanthine. Bioassays designed to measure exact Enzyme kinetic parameter Km value through establishing hyperbolic curve of an enzyme reaction with the use of exact values of cellular quantities for IC50 application under experimental conditions devised by presteady state approach for SSA validity.
Methods: Following saturation kinetic, the plot of hyperbolic equilibrium curve developed using initial rates of product formation as a function of (Si) through forward shift under circumstance dG0 the system allows product and reactant favored reactions in relation to (Ef) ≈ [E0 = KM] until complete saturation and estimates Km and Vmax of enzyme system under applied conditions. M-M equation used to assess experimental initial rate data for estimation of Km on excel using Solver and nonlinear least square coefficient correlation "R2" using logarithmic equation for nonlinear curve assessment.
Results: UV/Vis spectrophotometer selectively analyzed reacting components confirming Enzyme characteristic reaction constant Km equal toi15. 0 ± 2 μ mol acquired from the Hyperbolic curve developed through the use of exact (Si) ranges at selected parameter Km and Vmax. The curve assessed by Michaelis Menten equation provides Km value=14.99 μmol and non-linear least square coefficient correlation "R2" value equal to 0.9895, along with that optimized lysis buffer formulation. In the docked complexes, the interactive amino acids identified were MSE441, ALA 364, GLN363, MSE518, VAL362, GLY517, ASP538, ALA445, TYR521, and TYR444. 2D interactions revealed hydrophobic and alkyl interactions at the noncompetitive binding site of the enzyme and therefore recommended as potential inhibitors against 3ICS protein.
Conclusion: This study encourages biochemical analysis of the novel enzymes with the use of presteady state rationale in association with the computational tools as an effective way of designing drugs in a short time against selective enzymes to meet the current challenge efficiently.
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