Comparison of Resorufin Acetate andp-Nitrophenyl Acetate as Substrates for Chymotrypsin

Resorufin acetate is shown to be an attractive substrate to use with chymotrypsin since the absorbance of the product is several times more intense than that formed by the widely usedp-nitrophenyl acetate. Furthermore, under the right conditions, resorufin acetate allows convenient observation of the burst reaction by conventional spectrophotometry. The steady-statek_catvalues for chymotrypsin-catalyzed hydrolysis of resorufin acetate andp-nitrophenyl acetate are virtually the same, as expected for a rate-limiting deacylation step involving an identical intermediate from both substrates. Stopped-flow studies show that the maximal bursts of product from both substrates are again (in molar terms) about the same. When chymotrypsin is presented with a mixture of both substrates, the monitoring of reaction with resorufin acetate (at 571 nm) is not interfered with by simultaneous hydrolysis ofp-nitrophenyl acetate. Under these conditions,p-nitrophenyl acetate is shown to increase the burst rate constant for acylation of the enzyme by resorufin acetate, demonstrating unequivocally thatp-nitrophenyl acetate can bind to chymotrypsin elsewhere than in the active site.