Nucleophile in the active site of Escherichia coli galactose-1-phosphate uridylyltransferase: degradation of the uridylyl-enzyme intermediate to N3-phosphohistidine.
The [32P]uridylyl-enzyme intermediate form of Escherichia coli galactose-1-P uridylyltransferase can be converted to a [32P]phosphoryl-enzyme by first cleaving the ribosyl ring with NaIO4 and then heating at pH 10.5 and 50 degrees C for 1 h. After alkaline hydrolysis of the [32P]phosphoryl-enzyme the major radioactive product is N3-[32P]phosphohistidine. A lesser amount of 32Pi is also produced as a side product of the hydrolysis of N3-[32P]phosphohistidine. No N1-phosphohistidine, N-phospholysine, or phosphoarginine can be detected in these hydrolysates. It is concluded that the nucleophile in galactose-1-P uridylyltransferase to which the uridylyl group is bonded in the uridylyl-enzyme intermediate is imidazole N3 of a histidine residue. This degradation procedure should have general applicability in the degradation and characterization of nucleotidyl-proteins.
D055598:Chemical Phenomena; D002621:Chemistry; D003067:Coenzymes / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D009713:Nucleotidyltransferases / Q000032:analysis*; D010766:Phosphorylation; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase / Q000032:analysis*
D003067:Coenzymes; D006639:Histidine; D009713:Nucleotidyltransferases; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase