AMINO-ACID SUBSTITUTIONS IN THE CLEAVAGE SITE OF ACYL-COENZYME-A-ISOPENICILLIN-N-ACYLTRANSFERASE FROM PENICILLIUM-CHRYSOGENUM - EFFECT ON PROENZYME CLEAVAGE AND ACTIVITY

Site-directed mutagenesis of the penDE gene and expression in Escheric hia coli has produced recombinant acylcoenzyme A:isopenicillin N acylt ransferase (re-AT) containing amino-acid substitutions in the proenzym e cleavage site (1) region (Asp-Gly(102)down arrow Cys(103)-Thr-Thr). The effect of these substitutions on proenzyme cleavage and AT activit y has been investigated. The re-AT with substitutions at Cys(103) (Cys (103)-->Ser, Cys(103)-->Ala and Cys(103)-->Trp) were uncleaved and ina ctive. Substitutions at Asp(101) and Gly(102) (Asp(101)-->Gly, Gly(102 )-->Ala, Gly(102)-->Val, Gly(102)-->Met, Gly(102)-->Val and Asp(101)Gl y(102)-->GlyPhe) did not prevent proenzyme cleavage or abolish AT acti vity. Thr(105)-->Ser and Thr(105)-->Ala substitutions did not prevent proenzyme cleavage or AT activity; however, AT containing Thr(105)-->V al resulted in a significant inhibition of proenzyme cleavage.