Nature and environment of the sulfhydryls of membrane-associated D-lactate dehydrogenase of Escherichia coli.
Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid), has been used to titrate D-lactate dehydrogenase (D-LDH), a respiratory flavoenzyme of Escherichia coli. All six of the possible sulfhydryls titrate in the presence of 2% sodium dodecylsulfate, showing that D-lactate dehydrogenase does not contain any -S-S- bridges. In the native state, only two sulfhydryls are accessible in buffer and only one in the presence of lipid. Single-site mutations of each of the six cysteines of D-lactate dehydrogenase have been prepared. Each of the purified mutant proteins has full activity, demonstrating that an -SH group is not essential to the FAD-driven redox reaction. Ellman's titrations of the mutant proteins have led to the identification of cysteines 65, 146, 156, and 256 in the amino-terminal end as those containing the sulfhydryls that are not accessible in buffer or in buffer plus lipid. The cysteine at 422 is titrated only partially in buffer, while in buffer containing lipid, a necessary factor for full enzymatic activity, its sulfhydryl is inaccessible to the reagent. Cysteine 492 has been identified as containing the sulfhydryl that is accessible to the reagent under both conditions.
Dowd
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Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Pratt
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Sun
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Ho
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D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D003545:Cysteine / Q000032:analysis; D004220:Disulfides; D004228:Dithionitrobenzoic Acid; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009579:Nitrobenzoates; D012694:Serine; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013439:Sulfhydryl Reagents
D001425:Bacterial Outer Membrane Proteins; D004220:Disulfides; D009579:Nitrobenzoates; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C080134:n-octyl-5-dithio-2-nitrobenzoic acid; D012694:Serine; D004228:Dithionitrobenzoic Acid; D007770:L-Lactate Dehydrogenase; D003545:Cysteine