Purification and properties of a phenol sulphotransferase from Euglena using L-tyrosine as substrate

Abstract

A purification procedure based on (NH4)2SO4 precipitation, and chromatography on Affi-Gel Blue, DEAE-cellulose, hydroxyapatite and Bio-Gel P-60 yields a stable 6400-fold-purified active monomeric phenol (tyrosine) sulphotransferase of 26 kDa from W10BSmL, an aplastidic mutant of Euglena gracilis var. bacillaris. The apparent K(m) for adenosine 3'-phosphate 5'-phosphosulphate (PAPS) is 15 μM (60 μM tyrosine as substrate); adenosine 5'-phosphosulphate is inactive. L-Tyrosine gave the lowest apparent K(m) (33 μM) (with PAPS at 30 μM), but tyrosine esters, tyrosinamide, L-p-hydroxyphenylglycine and a number of tyrosine dipeptides were also active, with higher K(m) values. Nitrophenols (m- and p-) and chlorophenols (o-, m- and p-) were active, with higher Km values than for tyrosine. D-Tyrosine was inactive as a substrate, as was D-p-hydroxyphenylglycine and a number of other tyrosine derivatives lacking the carboxy carbonyl or the amino group, or having extra ring substituents or the hydroxy group in the wrong position. Adenosine 3',5'-bisphosphate and tyrosine O4-sulphate, products of the enzyme reaction with PAPS and tyrosine as substrates, showed competitive (K(i) = 20 μM and uncompetitive (K(i) = 500 μM) inhibition kinetics respectively. This appears to be the first phenol sulphotransferase to accept tyrosine as substrate. This membrane-bound enzyme may be involved in tyrosine transport as well as detoxification.