Purification and properties of a phenol sulphotransferase from Euglena using L-tyrosine as substrate.
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 Km for adenosine 3'-phosphate 5'-phosphosulphate (PAPS) is 15 microM (60 microM tyrosine as substrate); adenosine 5'-phosphosulphate is inactive. L-Tyrosine gave the lowest apparent Km (33 microM) (with PAPS at 30 microM), but tyrosine esters, tyrosinamide, L-p-hydroxyphenylglycine and a number of tyrosine dipeptides were also active, with higher Km 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 (Ki = 20 microM) and uncompetitive (Ki = 500 microM) 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.
Saidha
T
T
Biology Department, Brandeis University, Waltham, MA 02254.
Schiff
J A
JA
D000818:Animals; D015239:Arylsulfotransferase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004590:Electrophoresis, Paper; D005056:Euglena gracilis / Q000201:enzymology*; D007700:Kinetics; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism*
D014443:Tyrosine; D015239:Arylsulfotransferase