A microtiter plate assay for the characterization of serine proteases by their esterase activity.

The action of serine (and cysteine) proteases on peptide esters proceeds, as a generalization, orders of magnitude faster than the corresponding enzymatic hydrolysis of peptide bonds or peptide amides. Esterolysis liberates an alcohol while generating a free carboxyl group on the peptide; the proton produced can be detected by the use of an appropriate indicator. The action of trypsin on benzyloxycarbonylalanylarginine methyl ester was used as a model for the development of a simple microtiter plate assay procedure that takes advantage of the speed of these reactions and the ease of detection afforded by the color change of the indicator. A family of ester substrates of the form benzyloxycarbonylalanyl-X-methyl ester, in which X is one of the 20 common amino acids, was synthesized to allow the determination of the primary specificity profiles of serine proteases. Using a 96-well microtiter plate the specificity profiles of four enzymes with all 20 substrates can be carried out in approximately 4 h per enzyme, including setting up and data processing. The primary substrate preferences of trypsin, chymotrypsin, thrombin, pancreatic elastase, alpha-lytic protease, subtilisin, and proteinase K were determined to demonstrate the method and were found to be in good general agreement with reported specificities established by more conventional means.

Random mutagenesis of the substrate-binding site of a serine protease can generate enzymes with increased activities and altered primary specificities.

In the past, several point mutations have been introduced individually into the substrate-binding site of alpha-lytic protease (EC 3.4.21.12) and shown to affect its specificity in a predictable manner [Bone, R., Silen, J. L., & Agard, D. A. (1989) Nature 339, 191-195]. One of the resulting mutant enzymes (Met190Ala in the numbering system of Fujinaga et al.) [Fujinaga, M., Delbaere, L. T. J., Brayer, G. D., & James, M. N. G. (1985) J. Mol. Biol. 183, 479-502] cleaves at large hydrophobic residues. We chose this enzyme as the parent for a library of mutant proteases. The library was constructed by effecting combinatorial random substitution of up to four other residues (Gly191, Arg192, Met213, and Val218) thought likely to influence the primary specificity of the protease. Active enzymes in the library were screened with a range of synthetic substrates (encompassing 19 different amino acids in the P1 position) in order to evaluate their primary cleavage preferences. The amino acid sequences of active mutants revealed a strong preference for the replacement of Met213 with a His residue. This substitution also had the greatest observed effect on specificity, conferring a greatly increased and, in some cases, dominant ability to cleave at His residues in synthetic amide substrates. Mutant enzymes with greatly increased proteolytic activity were also found in the library.