THE THIOESTERASE-I OF ESCHERICHIA-COLI HAS ARYLESTERASE ACTIVITY AND SHOWS STEREOSPECIFICITY FOR PROTEASE SUBSTRATES

A thioesterase I gene was recloned and sequenced from Escherichia coli strain JM109. The overexpressed, matured enzyme from JM109 was purifi ed to homogeneity. The enzyme showed broad hydrolytic activity toward three kinds of substrates including acyl-CoAs, esters, and amino acid derivatives. The enzyme had a k(cat)/K-m, value of 0.363 s(-1)mu M(-1) , for a typical thioesterase I substrate, palmitoyl-CoA, The arylester ase activity of the enzyme was observed by its ability to hydrolyze se veral aromatic esters including cu-naphthyl acetate, alpha-naphthyl bu tyrate, phenyl acetate, benzyl acetate, and eight p-nitrophenyl esters . In kinetic studies a chymotrypsin-like substrate (an amino acid deri vative), N-carbobenzoxy-L-phenylalanine p-nitrophenyl ester (L-NBPNPE) , was the best substrate for the enzyme with a catalytic efficiency (k (cat)/K-m) of 4.00 s(-1)mu M(-1), which was 23 times higher than that of the enantiomer D-NBPNPE (0.171 s(-1)mu M(-1)). It was concluded tha t the thioesterase I of E. coli had arylesterase activity and it posse ssed stereospecificity for protease substrates. (C) 1997 Academic Pres s.