Km And Vmax Determination Of P-Npb Case Study

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Km and Vmax determination of glucocerebrosidase towards p-NPG
The Michaelis constant (Km) is a measure of the affinity of the enzyme towards the substrate, with smaller values representing greater affinity. Km and the maximum rate (Vmax) of leukocyte GCase were obtained through the Lineweaver-Burk plot (Figure 4) with artificial substrate p-NPG in concentrations from 0.71 mM to2.50 mM. Km and Vmax values for leukocyte GCase using p-NPG as substrate was found to be 12.6 mM and 333 U/mg respectively.

Fig. 4. Determination of Michaelis constant of GCase for different p-NPG substrate concentrations (0.71 mM-2.50 mM) in sodium acetate buffer (50mM, pH 5.0). All the samples were assayed in the presence of 6 mg/ml sodium taurocholate and 70 μl of
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Daniels, L.B., Glew, R.H., Diven, W.F., Lee, R.E., and Radin, N.S. (1981) An improved fluorometric leukocyte β-glucosidase assay for Gaucher’s disease. Clin. Chim. Acta, 115 (3), 369–375.
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Michelin, K., Wajner, A., Goulart, L. da S., Fachel, Â.A., Pereira, M.L.S., de Mello, A.S., Souza, F.T., Pires, R.F., Giugliani, R., and Coelho, J.C. (2004) Biochemical study on β-glucosidase in individuals with Gaucher’s disease and normal subjects. Clin. Chim. Acta, 343 (1), 145–153.
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