Galactose-1-phosphate uridylyltransferase: isolation and properties of a uridylyl-enzyme intermediate

Abstract

Galactose-1-P [phosphate] uridylytransferase [EC 2.7.7.12, from Escherichia coli] catalyzes the interconversion of UDP-glucose and galactose-1-P with UDP-galactose and glucose-1-P by a double displacement pathway involving a uridylyl-enzyme intermediate. The amount of radioactivity incorporated into the protein by uracil-labeled UDP-glucose is decreased by the presence of UDP-galactose, which competes with UDP-glucose for uridylylating the enzyme. The amount of glucose-1-P released upon reaction of the enzyme with UDP-glucose indicates that the dimeric enzyme contains > 1 active site/molecule, 1.7 on the average for the most active preparation obtained. This suggests that there is 1 uridylylation site/subunit and that the subunits are similar or identical. The uridylyl-enzyme is stable to milk alkaline conditions, 0.10 M NaOH at 60.degree. C for 1 h, but is very sensitive to acid, being largely hydrolyzed after 12 h at pH 3.5 and 4.degree. C. The principal radioactive product resulting from hydrolysis of [uracil-2-14C]uridylyl-enzyme under the latter conditions is [14C]UMP. The hydrolytic properties of the uridylyl-enzyme show that the uridylyl moiety is bonded to the protein through a phosphoramidate linkage. Complementary studies of the effects of group selective reagents on the activity of the enzyme suggest that the active site nucleophile to which the uridylyl group is bonded may be a histidine residue. The enzyme is rapidly inactivated by diethyl pyrocarbonate at pH 6 and 0.degree. C and reactivated by NH2OH.UDP-glucose at 0.5 mM fully protects the enzyme against diethyl pyrocarbonate, while 70 mM galactose-1-P has only a slight protective effect. Uridylyl-enzyme is inactivated by diethyl pyrocarbonate at no more than 2% of the rate for free enzyme. The enzyme is not inactivated by NaBH4 or by NaBH4 in the presence of UDP-glucose. It is not inhibited by 1 mM pyridoxal phosphate or by 0.5 mM 5-nitrosalicylaldehyde at pH 8.5, and it is not inactivated by NaBH4 in the presence of pyridoxal phosphate. The enzyme is inactivated by 5-50 .mu.M p-hydroxymercuribenzoate at pH 8.5, but substrates exert no detectable protective effect against this reagent. The enzyme apparently contains at least 1 essential SH group which is not located in the active site in such a way as to be shielded by substrates.