A. G. Cooper

The action of chymotrypsin on two new chromogenic substrates

Two new chromogenic substrates of chymotrypsin have been synthesized: N -succinyl- L -phenylalanine p -nitroanilide (SPNA) and N -glutaryl- L -phenylalanine p -nitroanilide (GPNA). Both are stable in the absence of enzyme and release p -nitroaniline (yellow) upon hydrolysis. GPNA is the more sensitive substrate being capable of quantifying less than 10 μg per milliliter chymotrypsin. The presence of Ca ++ decreases K m of the enzyme-substrate reaction but has a negligible effect upon k 3 . Na ++ , on the other hand, increases k 3 as well, and provides a suitable means of increasing the sensitivity of GPNA down to microgram levels of enzyme. In combination with an “all or none” assay the rate of hydrolysis of GPNA can be related to the molar concentration of enzyme. The reaction of chymotrypsin with GPNA is not subject to substrate activation over a 200-fold range of substrate concentration, nor is it affected by the presence of ethyl ammonium chloride. In these respects it differs from the reaction of trypsin with susceptible substrates.

Allosteric activation of chymotrypsin-catalyzed hydrolysis of specific substrates.

The rates of hydrolysis of three specific substrates of chymotrypsin, glutaryl-L-phenylalanine p-nitroanilide, acetyl-DL-tyrosine p-nitroanilide and acetyl-L-tyrosine anilide were enhanced by 2,2′-bis[α-(benzyldimethylammonium)methyl] azobenzene dibromide and less so by related compounds. Detailed studies with glutaryl-L-phenylalanine p-nitroanilide showed a 42-fold increase in kcat with no change in Km. No acceleration (or inhibition) was noted with esters, hydroxamides or proteins as substrates. Tryptic hydrolysis of benzoyl-DL-arginine p-nitroanilide was unaffected. It was concluded that certain quaternary compounds can act as allosteric effectors of chymotrypsin.