Biochemical characterisation of the enzyme responsible for "activated L-serine sulphydrase" activity in nematodes.
The biochemical properties of the enzyme responsible for nematode "activated L-serine sulphydrase" activity (L-cysteine + R-SH----cysteine thioether + H2S) have been investigated using primarily the gastro-intestinal nematodes Nippostrongylus brasiliensis and Haemonchus contortus. The activated L-serine sulphydrase enzyme was found to be cytosolic in origin and exhibited maximal activity at pH 9.0. Enzyme activity was widely distributed amongst the major tissues of adult female Ascaris suum but was particularly abundant in longitudinal muscle. The enzyme appeared to have a rigid specificity for L-cysteine as the primary thiol substrate, but was capable of utilising a number of sulphur amino acids (and derivatives) and nonphysiological thiols as second substrates. The best second thiol substrates were nonphysiological, hydroxyl-containing thiols that showed some structural similarity to the standard second substrate, 2-mercaptoethanol. Kinetic analyses revealed that the enzyme operates by a sequential catalytic mechanism, and the absolute Michaelis constants were: KL-cysteine = 0.21 +/- 0.02 mM and K2-mercaptoethanol = 5.58 +/- 0.59 mM. The enzyme was relatively insensitive to inhibition by a variety of substrate analogues and known inhibitors of pyridoxal 5'-phosphate dependent enzymes, whilst plant phenols caused significant levels of inhibition. The most potent inhibitors discovered were the anthelmintics bithionol, dichlorophene and hexachlorophene. Further characterisation revealed that hexachlorophene was a parabolic competitive inhibitor of the activated L-serine sulphydrase enzyme.
Walker
J
J
Department of Biological Sciences, University College of Wales, Aberystwyth, Dyfed, United Kingdom.
Barrett
J
J
D000818:Animals; D000871:Anthelmintics / Q000494:pharmacology; D003541:Cystathionine beta-Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003543:Cysteamine / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D006189:Haemonchus / Q000201:enzymology*; D006710:Homocysteine / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009348:Nematoda / Q000201:enzymology*; D009559:Nippostrongylus / Q000201:enzymology*; D010636:Phenols / Q000494:pharmacology; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D051381:Rats; D013379:Substrate Specificity
D000871:Anthelmintics; D010636:Phenols; D006710:Homocysteine; D003543:Cysteamine; D011732:Pyridoxal Phosphate; D003541:Cystathionine beta-Synthase; D003545:Cysteine