Purification, tandem mass characterization, and inhibition studies of oxidosqualene-lanosterol cyclase enzyme from bovine liver.
The oxidosqualene-lanosterol cyclase (OSC) from bovine liver has been isolated from the microsomal membrane fraction and purified to homogeneity by ultracentrifugation, Q-Sepharose, hydroxyapatite, and HiTrap heparin chromatographies. The purified protein required Triton X-100 to retain its highest activity. The cyclase had a molecular mass of approximately 70 and approximately 140 kDa, as evidenced by a single protein band on silver-stained SDS-PAGE and Coomassie-stained PAGE, respectively. Results from Edman degradation of OSC suggested that it might have a blocked N-terminus. Further peptide mapping coupled with tandem mass spectrometric determination identified three peptide fragments, ILGVGPDDPDLVR, LSAEEGPLVQSLR, and NPDGGFATYETK, which are highly homologous to human, rat, and mouse OSCs. The purified cyclase showed pH and temperature optima at pH 7.4 and 37 degrees C, respectively. The apparent K(M) and k(cat)/K(M) values were estimated to be 11 microM and 1.45 mM(-1)min(-1), respectively. Inhibition studies using both Ro48-8071 and N-(4-methylenebenzophenonyl)pyridinium bromide showed potent inhibition of OSC with an IC(50) of 11 nM and 0.79 microM, respectively. Results from DTNB modification and DTNB coupled with Ro48-8071 competition study suggest that two sulfhydryl groups are involved in the catalysis but not located in the substrate binding pocket or catalytic active site. The purified OSC was maximally inactivated by diethyl pyrocarbonate near neutral pH and re-activated by hydroxylamine, indicating the modification of histidine residues. The stoichiometry of histidine modification and the extent of inactivation showed that two essential histidine residues per active site are necessary for complete bovine liver OSC activity.
Wu
Tung-Kung
TK
Department of Biological Science and Technology, National Chiao Tung University, 300 Hsin-Chu, Taiwan, China. tkwmll@mail.nctu.edu.tw
Huang
Chiao-Yin
CY
Ko
Cheng-Yu
CY
Chang
Cheng-Hsiang
CH
Chen
Yu-Ju
YJ
Liao
Hsin-Kai
HK
D000595:Amino Acid Sequence; D000818:Animals; D001577:Benzophenones / Q000737:chemistry / Q000494:pharmacology; D002417:Cattle; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D019751:Intramolecular Transferases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D020539:Sequence Analysis, Protein / Q000379:methods; D017386:Sequence Homology, Amino Acid
D001577:Benzophenones; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D019811:Hydroxylamine; D006639:Histidine; D004228:Dithionitrobenzoic Acid; D019751:Intramolecular Transferases; C021924:lanosterol synthase; D003545:Cysteine; D004047:Diethyl Pyrocarbonate