Human placental sterylsulfatase. Interaction of the isolated enzyme with substrates, products, transition-state analogues, amino-acid modifiers and anion transport inhibitors.
The enzymatic properties of a homogeneous sterylsulfatase preparation isolated from human term placenta were studied. The enzyme exhibited both arylsulfatase and sterylsulfatase activity: it catalysed the hydrolysis of sulfuric acid esters of (in the order of decreasing specific activity) non-steroidal phenols, of a phenolic steroid, and of neutral 3 beta-, 21- and (though at a very low rate) 17 beta-hydroxysteroids. However, among all the substrates tested only the 3-sulfates of phenolic and neutral steroids exhibited high affinity towards the sulfatase. Vitamin D3 sulfate was not hydrolysed by the sterylsulfatase but strongly inhibited its activity. The products of the catalytic reaction, free steroids or phenols as well as the sulfate anion or analogues thereof, likewise interfered with the enzyme's activity. Ki values of unconjugated steroids were ten- to hundredfold higher than Km values of the respective sulfoconjugates. Inorganic sulfate only slightly inhibited the sulfatase activity; its inhibitory potency, however, increased in a time-dependent manner when it was preincubated with the enzyme prior to assay. In contrast to sulfate, the hypothetical transition-state analogues sulfite and vanadate acted as strong inhibitors of the sulfatase activity. According to the results of an analysis of the effect of pH on sterylsulfatase kinetics, enzyme constituents with pK values of approximately 5.8 and 8.0 are involved in a general acid-base catalysed reaction. Treatment of the sulfatase with amino-acid side chain modifying reagents directed against arginine, cysteine, cystine, serine or tyrosine residues did not result in significant alteration of its activity. Diethyl-pyrocarbonate known to react primarily with histidyl groups, however, rapidly inactivated the enzyme; this inactivation reaction was markedly retarded in the presence of substrate. Histidine thus appears to be essential for the catalytic activity of the sulfatase. Taken together, the present results reveal a considerable similarity between the catalytic mechanism of human placental sterylsulfatase and the ones already proposed for the lysosomal arylsulfatases A and B. Taurocholate, salicylate, ouabain, and 4,4'-substituted stilbene-2,2'-disulfonates are well known inhibitors of carrier-mediated transport of anions across cellular membranes. With the exception of ouabain, these compounds likewise turned out to inhibit the enzymatic hydrolysis of steryl sulfates; the pattern of dose dependences of their interference with the sulfatase activity resembles the one reported for inhibition of anion transport. Since the sterylsulfatase in vivo strongly is associated with cellular membranes including the plasma membrane of the syncytiotrophoblast, this finding supports the speculation that similar molecular structures may be involved in both placental transport and hydrolysis of anionic steryl sulfates.
Dibbelt
L
L
Institut für Biochemische Endokrinologie, Medizinischen Universität Lübeck.
Kuss
E
E
D000596:Amino Acids / Q000378:metabolism; D000838:Anions / Q000378:metabolism; D001192:Arylsulfatases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D003687:Dehydroepiandrosterone / Q000378:metabolism; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D013256:Steroids / Q000378:metabolism; D043266:Steryl-Sulfatase; D013462:Sulfur Radioisotopes
D000596:Amino Acids; D000838:Anions; D013256:Steroids; D013462:Sulfur Radioisotopes; D003687:Dehydroepiandrosterone; D001192:Arylsulfatases; D043266:Steryl-Sulfatase