Penicillase is an enzyme secreted by bacteria to inactivate the antibiotic penic

Question

Penicillase is an enzyme secreted by bacteria to inactivate the antibiotic penicillin. The enzyme has a molar mass of 30,000 and a single active site. The catalytic rate constant is 2000 s-1 and the Michaelis constant is KM = 5 x 10-5 M. In response to treatment with 5 microM of penicillin, a 1 mL (milliliter) suspension of bacterial cells secretes 0.04 micrograms of penicillase. Assuming that the enzyme quickly equilibrates with its substrate, what fraction of the enzyme will be complexed with penicillin in the early stages of the reaction?

Explanation / Answer

Ultrastructural localization of an extracellular protease in Pseudomonas fragi by using the peroxidase-antiperoxidase reaction; Thompson SS et al.; An extracellular protease, which previously has been found to correlate with the appearance of bleblike evaginations on the cell wall of Pseudomonas fragi ATCC 4973, was purified 38-fold by ammonium sulfate precipitation and Sephadex chromatography to yield a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Polyclonal rabbit antiserum raised against the purified enzyme had an enzyme-linked immunosorbent assay titer of 4 X 10(7) . The peroxidase antiperoxidase method was used to localize the neutral protease in P . fragi at the ultrastructural level . Electron microscopy of cell sections of this organism revealed that high concentrations of positive immunoperoxidase reaction product were located near the cell wall, whereas control sections stained with preimmune or heterologous serum did not show similar deposits to be present . These results are consistent with the hypothesis that blebs appearing on P . fragi contain high concentrations of neutral protease.

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