PURIFICATION, MOLECULAR CHARACTERIZATION AND CATALYTIC PROPERTIES OF A PSEUDOMONAS-FLUORESCENS ENZYME HAVING CHOLINESTERASE-LIKE ACTIVITY

An enzyme with a cholinesterase (ChE) activity, produced by Pseudomona s fluorescens, was purified to homogeneity in a three-step procedure. Analysis by non-denaturing and SDS-PAGE, and by isoelectric focusing, indicated that the enzyme was a monomer of 43 kDa, with a pi of 6.1. T he N-terminal sequence, AEPLKAVGAGEGQLDIVAWPGYIEA, showed some similar ities with proteins of the ChE family and a strong similarity with a p rotein from Escherichia coli with unknown structure and function. Chol inesterase activity at pH 7.0 and 25 degrees C was maximum with propio nylthiocholine as substrate (k(cat,app) = 670 min(-1)), followed by ac etylthiocholine, and significantly lower with butyrylthiocholine. Cata lytic specificity (k(cat)/K-m) was the same for propionylthiocholine a nd acetylthiocholine, but was two orders of magnitude lower for butyry lthiocholine. Kinetics of thiocholine ester hydrolysis showed inhibiti on by excess substrate which was ascribed to binding of a second subst rate molecule, leading to non-productive ternary complex (K-m = 35 mu M, K-SS = 0.49 mM with propionylthiocholine). There was low or no reac tivity with organophosphates and carbamates. The enzyme inhibited by e chothiophate (k(II) = 0.44 x 10(2) M-1 min(-1)) was not reactivated by pralidoxime methiodide. However, the P. fluorescens enzyme had affini ty for procainamide and decamethonium, two reversible ChE inhibitors u sed as affinity chromatography ligand and eluant, respectively. Althou gh similarity of the N-terminal amino acid sequence of the enzyme with an internal sequence of ChEs is weak, its catalytic activity towards thiocholine esters, and its affinity for positively charged ligands su pports the contention that this enzyme may belong to the ChE family. H owever, we cannot rule out that the enzyme belongs to another structur al family of proteins having cholinesterase-like properties. The react ion of the enzyme with organophosphates suggests that it is a serine e sterase, and currently this enzyme may be termed as having a cholinest erase-like activity. (C) 1998 Elsevier Science B.V. All rights reserve d.