A novel chiral microenvironmental probe at the active site of trypsin. Extrinsic cotton effects of acyl-trypsin possessing an enantiomeric pair of chromophores.

p-Amidinophenyl esters of an enantiomeric pair of N-(2,4-dinitrophenyl)alanine (N2Ph-Ala) were both efficiently hydrolyzed by trypsin. The acylation and deacylation rate constants for the D-isomer are 1/2.5 of those for the L-isomer. Slow rates of deacylation of the two substrates made it possible to prepare the pair of enantiomeric acyl-trypsins. Circular dichroic (CD) spectra of the purified acyl-trypsins revealed that the two extrinsic chromophores are somewhat differently oriented in the chiral environment of the active site, although both chromophores could couple intermolecularly with similar intrinsic chromophores near the active site. When p-amidinophenol was added, not only was the deacylation rate of N2Ph-DAla-trypsin noticeably increased, but also the transient CD spectrum of the enzyme derivative changed markedly in comparison with that of the L-derivative. The observations indicate that the two enantiomeric acyl groups at the active site are situated in different microenvironments.