Characterization of a multi-functional metal-mediated nuclease by MALDI-TOF mass spectrometry.
Mass spectrometric analysis of reaction products allows simultaneous characterization of activities mediated by multifunctional enzymes. By use of MALDI-TOF mass spectrometry, the relative influence of magnesium and manganese promoted exonuclease and phosphatase activities of Esherichia coli exonuclease III have been quantitatively measured, offering a rapid and sensitive alternative to radioactivity quantification and gel electrophoresis procedures for determination of reaction rate constants. Manganese is found to promote higher levels of exonuclease activity, which could be a source of mutagenic effects if this ion were selected as the natural cofactor. Several potential applications of these methods to quantitative studies of DNA repair chemistry are also described.
Puapaiboon
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Evans Laboratory of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, OH 43210, USA.
Jai-Nhuknan
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Cowan
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JA
D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D005092:Exonucleases / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000494:pharmacology; D008345:Manganese / Q000737:chemistry / Q000494:pharmacology; D008670:Metals / Q000737:chemistry* / Q000494:pharmacology; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000187:drug effects / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D008670:Metals; D009841:Oligonucleotides; D008345:Manganese; D005090:Exodeoxyribonucleases; D005092:Exonucleases; C020133:exodeoxyribonuclease III; D010744:Phosphoric Monoester Hydrolases; D008274:Magnesium