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Several azasugar and glycal inhibitors were studied to map the active site of α-L-fucosidase enzyme.

A recombinant human Lewis α(1,3/1,4)fucosyltranferase has been studied for its acceptor substrate specificity and used in the synthesis of sialyl Lex and derivatives.

The pH rate profile for the hydrolysis of diethyl-p-nitrophenyl phosphate catalyzed by the phosphotriesterase from Pseudomonas diminuta shows a requirement for the deprotonation of an ionizable group for full catalytic activity. This functional group has an apparent pKa of 6.1 +/- 0.1 at 25 degrees C, delta Hion of 7.9 kcal/mol, and delta Sion of -1.4 cal/K.mol. The enzyme is not inactivated in the presence of the chemical modification reagents dithiobis-(2-nitrobenzoate), methyl methane thiosulfonate, carbodiimide, pyridoxal, butanedione, or iodoacetic acid and thus cysteine, asparate, glutamate, lysine, and arginine do not appear to be critical for catalytic activity. However, the phosphotriesterase is inactivated completely with methylene blue, Rose Bengal, or diethyl pyrocarbonate. The enzyme is not inactivated by diethyl pyrocarbonate in the presence of bound substrate analogs, and inactivation with diethyl pyrocarbonate is reversible upon addition of neutralized hydroxylamine. The modification of a single histidine residue by diethyl pyrocarbonate, as shown by spectrophotometric analysis, is responsible for the loss of catalytic activity. The pKinact for diethyl pyrocarbonate modification is 6.1 +/- 0.1 at 25 degrees C. These results have been interpreted to suggest that a histidine residue at the active site of phosphotriesterase is facilitating the reaction by general base catalysis.

The phosphotriesterase produced from the opd cistron of Pseudomonas diminuta was purified 1500-fold to homogeneity using a combination of gel filtration, ion exchange, hydrophobic, and dye matrix chromatographic steps. This is the first organophosphate triesterase or organophosphofluoridate hydrolyzing enzyme to be purified to homogeneity. The enzyme is a monomeric, spherical protein having a molecular weight of 39,000. A single zinc atom is bound to the enzyme and is required for catalytic activity. Incubation with metal chelating compounds, o-phenanthroline, EDTA, or 2,6-pyridine dicarboxylate inactivate the enzyme. The kinetic rate constants, kcat and kcat/Km, for the hydrolysis of paraoxon are 2100 s-1 and 4 x 10(7) M-1 s-1, respectively. The enzyme is inhibited competitively by dithiothreitol, dithioerithritol, and beta-mercaptoethanol. In addition to paraoxon the phosphotriesterase was found to hydrolyze the commonly used organophosphorus insecticides, dursban, parathion, coumaphos, diazinon, fensulfothion, methyl parathion, and cyanophos.