Galactose l-Phosphate Uridylyltransferase of Escherichia coli

Saito, Shigeru; Ozutsumi, Michie; Kurahashi, Kiyoshi

doi:10.1016/s0021-9258(18)95970-3

Cited by 44 publications

(20 citation statements)

References 25 publications

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“…Enzymes. Galactose-1-P uridylyltransferase was purified from a regulatory mutant of E. coli, ATCC-27797, by a modification of the published procedure (Saito et al, 1967).…”

Section: Methodsmentioning

confidence: 99%

“…We eliminated the addition of streptomycin sulfate to the crude extract because we did not observe precipitate formation in our extracts and the addition proved to be unnecessary. We also modified the calcium phosphate gel treatment, step 4 of the published procedure (Saito et al, 1967). The first addition of calcium phosphate gel was carried through as described to remove inactive protein, but the second addition, to adsorb the enzyme, was eliminated.…”

Section: Methodsmentioning

confidence: 99%

“…The calcium phosphate gel adsorption and gel-cellulose chromatography effected a sixfold increase in specific activity. The balance of the purification was followed essentially as described by Saito et al (1967) for a number of preparations. Recently we further modified the procedure by eliminating DEAE-cellulose column chromatography and proceeding directly to the hydroxylapatite column chromatography as described (Saito et al, 1967).…”

Section: Methodsmentioning

confidence: 99%

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

Wong¹,

Sheu²,

Lee³

et al. 1977

Biochemistry

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Galactose-1-P uridylyltransferase catalyzes the interconversion of UDP-galactose 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 completes 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 more than one active site per molecule, 1.7 on the average for the most active preparation obtained. This suggests that there is one uridylylation site per subunit and that the subunits are similar or identical. The ureidylyl-enzyme is stable to mild alkaline conditions, 0.10 M NaOH at 60 degrees C for 1 h, but is very sensitive to acid, being largely hydrolyzed after 12 h at pH 3.5 and 4 degrees C. The principal radioactive product resulting from hydrolysis of [uracil-2-14C]uridylyl-ens of the uridylyl-enzyme under the latter conditions is [l]ump. The hydrolytic properties of the uridylyl-enzyme show that the uridylyl moiety is bonded to the protein through a phosphoramidate linkage. Complementary studies on 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 degrees 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 in 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 8.6 and it is not inactivated by NaBH4 in the presence of pyridoxal phosphate. The enzyme is inactivated by 5 to 50 muM p-hydroxymercuribenzoate at pH 8.5, but substrates exert no detectable protective effect against this reagent. It is concluded that the enzyme contains at least one essential sulfhydryl group which is not located in the active site in such a way as to be shielded by substrates.

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“…Enzymes. Galactose-1-P uridylyltransferase was purified from a regulatory mutant of E. coli, ATCC-27797, by a modification of the published procedure (Saito et al, 1967).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

Wong¹,

Sheu²,

Lee³

et al. 1977

Biochemistry

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“…The amino acid sequence of the uridylyltransferase is and humans share approximately 39% and 46% sequence identity with the enzyme from E. coli; these enzymes exist as a.2 dimers (Saito et al, 1967;Segawa & Fukasawa, 1979;Dale & Popjak, 1976). The dimeric enzyme from E. coli has a molecular mass of 80 kDa, with 348 amino acids per subunit (Lemaire & Mueller-Hill, 1986;Cornwell et al, 1987).…”

mentioning

confidence: 99%

Three-Dimensional Structure of Galactose-1-phosphate Uridylyltransferase from Escherichia coli at 1.8 .ANG. Resolution

Wedekind¹,

Frey²,

Rayment³

1995

Biochemistry

120

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Galactose-1-phosphate uridylyltransferase catalyzes the reversible transfer of the uridine 5'-monophosphoryl moiety of UDP-glucose to the phosphate group of galactose 1-phosphate to form UDP-galactose. This enzyme participates in the Leloir pathway of galactose metabolism, and its absence is the primary cause of the potentially lethal disease galactosemia. The three-dimensional structure of the dimeric enzyme from Escherichia coli complexed with uridine 5'-diphosphate is reported here. The structure was solved by multiple isomorphous replacement and electron density modification techniques and has been refined to 1.8 A resolution. Enzyme subunits consist of a single domain with the topology of a "half-barrel". The barrel staves are formed by nine strands of antiparallel beta-sheet. The barrel axis is approximately parallel to the local dyad that relates each subunit. Two amphipathic helices fill the half-barrel sequestering its hydrophobic interior. An iron atom resides on the outside of the barrel, centered in the subunit interface. Intrasubunit coordination to iron resembles a distorted square pyramid formed by the equatorial ligation of two histidines and a bidentate carboxylate group and a single axial histidine. The subunit interface is stabilized by this coordination and is further characterized by the formation of two intermolecular "mini-sheets" distinct from the strands of the half-barrel. Loops that connect the mini-sheet strands contribute to the formation of the active site, which resides on the external surface of the barrel rim. Loops of the barrel strands are tethered together by a structural zinc atom that orients the local fold in a manner essential for catalysis. In one of the latter loops, S gamma of a cysteine is modified by beta-mercaptoethanol, which prevents the alpha-phosphorus of the nucleotide from access to the nucleophile His166. This conformation does not appear to perturb the interactions to the uracil and ribose moieties as mediated through the side chains of Leu54, Ohe75, Asn77, Asp78, Phe79, and Val108. Several of the latter residues have been implicated in human galactosemia. The present structure explains the deleterious effects of many of those mutations.

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“…The catalytic mechanism includes tbe reversible oxidation of UDPgalactose or UDP-glucose to UDP-4-ketoglucose and concomitant reduction of NAD+ to NADH at the active site. Galactose-l-P uridylyltransferase from E. coli consists of two identical subunits and has an overall molecular weight of 80 000 (9). The enzymatic mechanism is a double-displacement of the uridylyl group that obeys ping-pong kinetics and requires the formation of a covalent uridylyl-enzyme as the intermediate (10).…”

mentioning

confidence: 99%

Preparation and characterization of a bifunctional fusion enzyme composed of UDP-galactose 4-epimerase and galactose-1-P uridylyltransferase

Tamada¹,

Swanson²,

Arabshahi³

et al. 1994

Bioconjugate Chem.

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A fusion enzyme consisting of UDP-galactose 4-epimerase and galactose-1-P uridylyltransferase with an intervening Ala3 linker was constructed by in-frame fusion of E. coli gene galT to the 3'-terminus of the E. coli gene galE that had been extended with the coding sequence for three alanine residues, all contained within a high-expression plasmid. The fusion enzyme was expressed in E. coli and purified 24-fold to about 98% homogeneity by chromatography on hydroxylapatite and Q-Sepharose. On the basis of the comparison of the elution profile for enzyme activities upon gel permeation chromatography (Sephacryl S-400) with the molecular weight of 80,000 determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the fusion enzyme appears to exist in monomeric, dimeric, and tetrameric forms, all of which exhibit both enzymatic activities. The Km values of the fusion enzyme for substrates were similar to those for the corresponding native enzymes, except for UDP-glucose, but the kcat values were smaller than those for the native enzymes. The fusion enzyme shows kinetic advantages in that the initial velocity to produce glucose-1-P from UDP-galactose and galactose-1-P is about 20% faster than that for a mixture of equal activities of the separate enzymes.

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Galactose l-Phosphate Uridylyltransferase of Escherichia coli

Cited by 44 publications

References 25 publications

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

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

Three-Dimensional Structure of Galactose-1-phosphate Uridylyltransferase from Escherichia coli at 1.8 .ANG. Resolution

Preparation and characterization of a bifunctional fusion enzyme composed of UDP-galactose 4-epimerase and galactose-1-P uridylyltransferase

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