Problem Studies of a novel endopeptidase

What reaction is catalysed by an endopeptidase, as opposed to an exopeptidase?

A new enzyme, of bacterial origin, is being studied for its potential use in a washing powder preparation. The enzyme is an endopeptidase, and has been purified by a variety of chromatographic techniques.

The activity of the enzyme has been determined using an assay based on the hydrolysis of a synthetic substrate, _p-nitrophenyl acetate. On hydrolysis this (colourless) substrate yields 1 mol of _p-nitrophenol (which is yellow) for each mole of substrate hydrolysed.

In the following experiments, 0.1 mL of a solution containing 1 mg of the purified protein per litre was used in each incubation. The enzyme was incubated at 30 °C and pH 7.5, for 10 min; the formation of ^-nitrophenol was followed spectro-photometrically. The results are shown in Table 2.5.

Using these results, determine the Vmax of the enzyme under these incubation conditions.

Given the relative molecular mass of the enzyme (= 50,000), calculate the catalytic rate constant, kcat (the maximum rate of reaction expressed in mole of product formed per mole of enzyme per second).

Table 2.5 The rate of reaction of a novel endopeptidase incubated with p-nitrophenyl acetate as a model substrate

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