A promiscuous glutathione transferase transformed into a selective thiolester hydrolase

Human glutathione transferase A1-1 (hGST A1-1) can be reengineered by rational design into a catalyst for thiolester hydrolysis with a catalytic proficiency of 1.4 x 10(7) M(-1). The thiolester hydrolase, A216H that was obtained by the introduction of a single histidine residue at position 216 catalyzed the hydrolysis of a substrate termed GSB, a thiolester of glutathione and benzoic acid. Here we investigate the substrate requirements of this designed enzyme by screening a thiolester library. We found that only two thiolesters out of 18 were substrates for A216H. The A216H-catalyzed hydrolysis of GS-2 (thiolester of glutathione and naphthalenecarboxylic acid) exhibits a k(cat) of 0.0032 min(-1) and a KM of 41 microM. The previously reported catalysis of GSB has a k(cat) of 0.00078 min(-1) and KM of 5 microM. The k(cat) for A216H-catalyzed hydrolysis of GS-2 is thus 4.1 times higher than for GSB. The catalytic proficiency (k(cat)/KM)/k(uncat) for GS-2 is 3 x 10(6) M(-1). The promiscuous feature of the wt protein towards a range of different substrates has not been conserved in A216H but we have obtained a selective enzyme with high demands on the substrate.     

A simple model for the water o-H2 complex

The infrared spectrum of the complex between o-H2 and H2O, D2O, or HDO, isolated in a matrix of solid p-H2, has been studied between 20 and 4500 cm(-1). In addition the infrared spectrum of the complex between p-D2 and H2O in solid o-D2 has been studied. The spectral shifts are interpreted as the result of the quadrupole-dipole interaction between hydrogen and water.     

ESR applications to photochemical polymer reactions

ESR spectroscopy combined with spin-trapping technique is a selective and powerful method for studies of reaction mechanisms and intermediates in free radical reactions of polymer systems. The method has been applied to several problems in our research program of which three are presented in this paper. - The mechanism of photo-initiation with aromatic ketone/tert. amine and subsequent photocuring of acrylic coatings has been largely resolved. - The mechanism of photocrosslinking of ethylene-propylenediene copolymers (EPDM elastomers) studied by model compounds has been shown to involve formation of allyl radicals in the diene groups and crosslinking by allyl radical combination. - The photoinitiated crosslinking of polyethylenes (LD, LLD and HD) has been studied using model compounds. It involves formation of primary alkyl and allyl radicals which crosslink the polymer by radical combination. - The mechanism of photocrosslinking indicates a second order reaction, i.e. rate of crosslinking depends of the square of irradiation intensity which has been verified experimentally. This result is the basis for development of rapid crosslinking processes of technical and industrial interest.