Fluorescent analogs of UDP-glucose and their use in characterizing substrate binding by toxin A from Clostridium difficile.
Uridine-5'-diphospho-1-alpha-d-glucose (UDP-Glc) is a common substrate used by glucosyltransferases, including certain bacterial toxins such as Toxins A and B from Clostridium difficile. Fluorescent analogs of UDP-Glc have been prepared for use in our studies of the clostridial toxins. These compounds are related to the methylanthraniloyl-ATP compounds commonly used to probe the chemistry of ATP-dependent enzymes. The reaction of excess methylisatoic anhydride with UDP-Glc in aqueous solution yields primarily the 2' and 3' isomers of methylanthraniloyl-UDP-Glc (MUG). As the 2' and 3' isomers readily interconvert, this isomeric mixture was copurified by HPLC away from the other isomeric products, and was characterized by a combination of NMR, fluorescence and mass spectrometric methods. TcdA binds MUG competitively with respect to UDP-Glc with an affinity of 15 +/- 2 microm in the absence of Mg2+. There is currently no evidence that the fluorescent substrate analog is turned over by the toxin in either glucosyltransferase or glucosylhydrolase reactions. Using a competition assay, the affinity of UDP-Glc was determined to be 45+/-10 microm in the absence of Mg2+. The binding of UDP-Glc and Mg2+ are highly coupled with Mg2+ affinities in the range of 90-600 microm, depending on the experimental conditions. These results imply that one of the significant roles of the metal ion might be to stabilize the enzyme-substrate complex prior to initiation of the transferase chemistry.
Department of Chemistry, Indiana University, Bloomington, IN 47405, USA.
Sudeep
S
Bhattacharyya
Amy
A
Kerzmann
Andrew L
AL
Feig
D001427:Bacterial Toxins / Q000378:metabolism*; D001667:Binding, Competitive; D020134:Catalytic Domain; D016360:Clostridioides difficile / Q000378:metabolism*; D004768:Enterotoxins / Q000378:metabolism*; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000378:metabolism*; D007536:Isomerism; D008274:Magnesium / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014532:Uridine Diphosphate Glucose / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism*; D062367:ortho-Aminobenzoates / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism*
D001427:Bacterial Toxins; D004768:Enterotoxins; D005456:Fluorescent Dyes; C465221:methylanthraniloyl-UDP-glucose; D062367:ortho-Aminobenzoates; C041348:tcdA protein, Clostridium difficile; D008274:Magnesium; D014532:Uridine Diphosphate Glucose