ELIMINATION OF THE SENSITIVITY OF L-ASPARTASE TO ACTIVE-SITE-DIRECTEDINACTIVATION WITHOUT ALTERATION OF CATALYTIC ACTIVITY

The catalytic activity of the enzyme L-aspartase from Escherichia coli has previously been shown to be sensitive to sulfhydryl reagents. The use of group-specific reagents, and a sequence homology comparison st udy among the fumarase-aspartase family of enzymes, has not, however, lead to the identification of a specific, essential cysteinyl residue. We have recently shown that L-aspartate-beta-semialdehyde is an alter native substrate for L-aspartase, producing fumaric acid semialdehyde (FAA) which specifically inactivates the enzyme [Schindler, J. F., & V iola, R. E. (1994) Biochemistry 33, 9365]. Proteolytic digests of the resulting inactivated enzyme have now been mapped by HPLC and mass spe ctrometry. A specific residue (Cys-273) has been determined to be the site of FAA modification. Site-directed mutagenesis of this cysteine i n the E. coli enzyme has produced altered enzymes which are considerab ly less sensitive to active-site-directed inactivation, while retainin g full catalytic activity. Thus, cysteine-273 has been identified as a n active-site nucleophile that, while not directly involved in catalys is in L-aspartase, is poised to attack an activated double bond in an enzyme-bound product analogue.