Purification and characterization of human hepatic cysteine-conjugate beta-lyase.
Cysteine-conjugate beta-lyase (EC 4.4.1.13) was purified about 880-fold from human liver obtained post mortem. The purification procedure included (NH4)2SO4 precipitation, chromatography on DEAE-cellulose and hydroxyapatite, gel filtration on Sephadex G-200, and chromatofocusing. The purified enzyme cleaves the C-S bond of several S-aryl-L-cysteines to yield equimolar amounts of thiols, pyruvic acid and ammonia via an alpha beta-elimination reaction. The Mr of the enzyme was estimated to be 88,000 by gel filtration. The enzyme is thermolabile, has a pH optimum of 8.5, and an apparent Km of 0.7 mM towards S-(p-bromophenyl)-L-cysteine. The enzyme requires pyridoxal 5'-phosphate as a cofactor, and hence the enzyme activity was completely abolished by hydroxylamine. No effect of EDTA or thiol-blocking reagents was observed on the activity of the enzyme.
D013437:Carbon-Sulfur Lyases; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008190:Lyases / Q000302:isolation & purification / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
D011732:Pyridoxal Phosphate; D008190:Lyases; D013437:Carbon-Sulfur Lyases; C019969:S-alkylcysteine lyase; D003545:Cysteine