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Enzymatic Characterization of Aldose Reductase and Its Inhibitors
- Publication Year :
- 2016
-
Abstract
- Human aldose reductase (hAR) is a NADPH-dependent oxidoreductase that reduces aldehydes to their corresponding alcohols with the oxidation of NADPH. It has been shown that two forms of this enzyme can occur. When Cysteine-298 in the active site is mutated, different kinetic variables are experienced that correlate to the form of enzyme being obtained. Mutant C298A kinetic studies showed that the Michaelis-Menten constant, Km, had an 18-fold increase, the turnover number, kcat, decreased about 83%, and the catalytic efficency, kcat/Km, decreased about 100% for the mutant enzyme compared to the wildtype for the forward reaction using DL-glyceraldehyde as substrate and NADPH as cofactor. Mutant C298A kinetic studies showed that the Michaelis-Menten constant, Km, had an 4-fold decrease, the turnover number, kcat, decreased about 60%, and the catalytic efficency, kcat/Km, increased about 39% for the mutant enzyme compared to the wildtype for the reverse reaction using benzyl alcohol as substrate and NADP+ as cofactor. The mutation proved beneficial for the enzyme, shown to have been the targeted native form of the enzyme.Thermal analysis of clofibric acid samples in sodium chloride mixtures of 10% (w/w) ratios were investigated by thermogravimetric analysis and mass spectroscopy. Kinetic parameters like activation energy Ea, pre-exponential factor A, and order of reaction n were calculated for each sample's decomposition. Trends within the kinetic parameters occurred with increase of clofibric acid percentage. Mass spectroscopy data shows a detection of CO2 with decomposition of clofibric acid samples. This data can provide insight on the possible mechanisms in clofibric acid decomposition.
Details
- Language :
- English
- Database :
- OpenDissertations
- Publication Type :
- Dissertation/ Thesis
- Accession number :
- ddu.oai.etd.ohiolink.edu.ysu1472069987