Final answer:
The experiment examines the Michaelis-Menten kinetics of beta-galactosidase using a CelB-GFP fusion protein. It determines Vmax and Km through experiments with varied substrate and enzyme concentrations, and assesses enzyme behavior using fluorescence measurements.
Step-by-step explanation:
Michaelis-Menten Kinetics of Beta-Galactosidase
The experiment concerning the Michaelis-Menten kinetics of beta-galactosidase activity involves the use of a CelB-GFP fusion protein. This enzyme, involved in lactose digestion in E. coli, exhibits kinetics that can be graphically represented as a rectangular hyperbola as described by Leonor Michaelis and Maud Menten. In analyzing enzymes like beta-galactosidase, kinetic parameters such as Vmax (maximum reaction rate) and Km (Michaelis constant) can be deduced using linear regression of double-reciprocal plots, further informing on the enzyme's efficiency and affinity for the substrate.
Experimental conditions may include varying substrate concentrations typically ranging from 1 to approximately 200 µM, with enzyme concentrations adjusted accordingly. This ensures that reaction rates fall within a measurable range, from 0.1 to about 5 uM/min. Fluorescence measurements in these experiments are important for determining enzyme activity and are taken using semi-micro cuvettes to minimize inner filter effects.
Such biochemical experiments are significant not only for understanding enzyme kinetics but also for exploring the regulation of gene expression, as seen in mutants of E. coli that show different capacities to grow on lactose based on the availability and activity of the necessary enzymes encoded within the lac operon. These experiments provide insights into how enzymes behave under cellular conditions and how they may respond to various regulatory or inhibitory substances.