Cysteine reactivity in Thermoanaerobacter brockii alcohol dehydrogenase.
The free cysteine residues in the extremely thermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized using selective chemical modification with the stable nitroxyl biradical bis(1-oxy-2,2,5,5-tetramethyl-3-imidazoline-4-yl)disulfide, via a thiol-disulfide exchange reaction and with 2[14C]iodoacetic acid, via S-alkylation. The respective reactions were monitored by electron paramagenetic resonance (EPR) and by the incorporation of the radioactive label. In native TBADH, the rapid modification of one cysteine residue per subunit by the biradical and the concomitant loss of catalytic activity was reversed by DTT. NADP protected the enzyme from both modification and inactivation by the biradical. RPLC fingerprint analysis of reduced and S-carboxymethylated lysyl peptides from the radioactive alkylated enzyme identified Cys 203 as the readily modified residue. A second cysteine residue was rapidly modified with both modification reagents when the catalytic zinc was removed from the enzyme by o-phenanthroline. This cysteine residue, which could serve as a putative ligand to the active-site zinc atom, was identified as Cys 37 in RPLC. The EPR data suggested a distance of < or 10 A between Cys 37 and Cys 203. Although Cys 283 and Cys 295 were buried within the protein core and were not accessible for chemical modification, the two residues were oxidized to cystine when TBADH was heated at 75 degrees C, forming a disulfide bridge that was not present in the native enzyme, without affecting either enzymatic activity or thermal stability. The status of these cysteine residues was verified by site directed mutagenesis.
Peretz
M
M
Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel.
Weiner
L M
LM
Burstein
Y
Y
D000426:Alcohol Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D001421:Bacteria, Anaerobic / Q000201:enzymology*; D003545:Cysteine; D004220:Disulfides / Q000032:analysis; D004229:Dithiothreitol / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D016988:Gram-Positive Asporogenous Rods, Irregular / Q000201:enzymology*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007461:Iodoacetates / Q000378:metabolism; D019807:Iodoacetic Acid; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013113:Spin Labels; D013816:Thermodynamics
D004220:Disulfides; D006146:Guanidines; D007461:Iodoacetates; D011994:Recombinant Proteins; D013113:Spin Labels; D000426:Alcohol Dehydrogenase; D019791:Guanidine; D003545:Cysteine; D004229:Dithiothreitol; D019807:Iodoacetic Acid