VIBRIO-MIMICUS ARYLESTERASE HAS THIOESTERASE AND CHYMOTRYPSIN-LIKE ACTIVITY

A Vibrio mimicus serine arylesterase and an Escherichia coli thioester ase/serine protease share 49.4% amino acid identity. The arylesterase has thioesterase activity for benzoyl-CoA and chymotrypsin-like activi ty for N-carbobenzoxy-L-phenylalanine p-nitrophenyl ester (NBPNPE). Th e gene encoding the V. mimicus enzyme is designated etpA. Substituting Ser31 of the V. mimicus enzyme with a glycine or an alanine altered i ts activity. In comparison with wild type enzyme, the S31A enzyme show ed a 5-fold increase and 57% decrease in the catalytic efficiency for benzoyl-CoA and NBPNPE, respectively, and the S31G enzyme showed a 3.6 -fold increase and 43% decrease in the catalytic efficiency for benzoy l-CoA and NBPNPE, respectively. For the two mutant enzymes an 8-fold d ecrease and a 6- to 7-fold increase in K-m were seen for benzoyl-CoA a nd NBPNPE, respectively. The mutagenesis results prove that residue 31 plays an important role in the substrate-specificity. (C) 1995 Academ ic Press, Inc.