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 °C for 1 h. After alkaline hydrolysis of the [32P]phosphoryl-enzyme the major radioactive product is N3-33P]-phosphohistidine. A lesser amount of 32P1 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. © 1979, American Chemical Society. All rights reserved.