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E317A
relative activity of mutant protein: 0.1% (compared to wild-type 100%)
E317A/E317Q
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site-directed mutagenesis, substrate binding compared to wild-type
E317C
relative activity of mutant protein: 0.8% (compared to wild-type 100%)
E317D
relative activity of mutant protein: 0.4% (compared to wild-type 100%)
E317H
relative activity of mutant protein: 0.1% (compared to wild-type 100%)
H280A/A281A/A282A
mutant AAA, 20 or 200 mM lactose as acceptor, 14 and 41% galactosyltransferase activity (UDP-galactose as donor), 4 and 6% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0
H280A/A281G/A282G
mutant AGG, 20 or 200 mM lactose as acceptor, 0.4 and 1% galactosyltransferase activity (UDP-galactose as donor), 0.3 and 2% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0, among the mutants with highest N-acetylgalactosamnine transferase activity and ability to transfer C2-modified sugars
H280A/A281G/A282G/L283I
AGGI-mutant, instable, leucin 283 is important for enzyme stability
H280A/A281G/A282G/L283L
AGGL-mutant, higher N-acetylgalactosamine transferase activity than galactosyltransferase activity, leucin 283 is important for enzyme stability
H280A/A281G/A282G/L283V
AGGV-mutant, instable, leucin 283 is important for enzyme stability
H280G/A281A/A282A
protein unstable, precipitating during purification
H280G/A281A/A282G
mutant GAG, 20 or 200 mM lactose as acceptor, 5 and 27% galactosyltransferase activity (UDP-galactose as donor), 4 and 6% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0
H280G/A281G/A282G/L283I
GGGI-mutant, instable, leucin 283 is important for enzyme stability
H280G/A281G/A282G/L283L
GGGL-mutant, higher N-acetylgalactosamine transferase activity than galactosyltransferase activity, leucin 283 is important for enzyme stability
H280G/A281G/A282G/L283V
GGGV-mutant, instable, leucin 283 is important for enzyme stability
H280L/A281G/A282G
mutant LGG, 20 or 200 mM lactose as acceptor, 2 and 1% galactosyltransferase activity (UDP-galactose as donor), 3 and 2% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0
H280Q
80fold reduction in catalytic activity
H280R/A281A/A282A/L283I
RAAI-mutant, no catalytic activity, crystallisation
H280S/A281G/A282G
mutant SGG, 20 or 200 mM lactose as acceptor, 5 and 5% galactosyltransferase activity (UDP-galactose as donor), 2 and 11% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0, among the mutants with highest GalNac transferase activity and ability to transfer C2-modified sugars
H280T/A281G/A282G
mutant TGG, 20 or 200 mM lactose as acceptor, 3 and 15% galactosyltransferase activity (UDP-galactose as donor), 1 and 6% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0
H280V/A281G/A282G
mutant VGG, 20 or 200 mM lactose as acceptor, 4 and 16% galactosyltransferase activity (UDP-galactose as donor), 0.5 and 3% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0
H315Q
mutant protein shows modest changes in kinetic parameters for lactose
H315R
mutant shows a major losss in catalytic activity arising from a 500fold reduction in kcat
H319A
mutant shows a 2fold reduction for kcat
Q247E
50fold reduction in turnover number for transferase reaction, unchanged hydrolase activity, Km-values for UDP-galactose and lactose are similar to the wild-type values
R365K
mutation reduces turnover-number for UDP-galactose hydrolysis about 10fold but has no significant effect on the affinity for UDP-galactose
S318A
mutant shows a 10fold reduction for kcat
W249G
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1.95fold increase in Km-value for UDP-galactose in galactosyltransferase reaction, turnover number of hydrolase reaction is identical to wild-type value, 2.1fold increase in Km-value for UDP-galactose in hydrolase reaction. Mutation does not affect the overall structure of the enzyme or its interactions with ligands
W250F
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2.6fold increase in turnover number of galactosyltransferase reaction, 9.3fold increase in Km-value for UDP-galactose in galactosyltransferase reaction, 1.1fold decrease in Km-value for lactose, 1.4fold increase in turnover number of hydrolase reaction, 1.5fold increase in Km-value for UDP-galactose in hydrolase reaction
W250Y
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1.2fold increase in turnover number of galactosyltransferase reaction, 5.2fold increase in Km-value for UDP-galactose in galactosyltransferase reaction, 2fold decrease in Km-value for lactose, 1.8fold increase in turnover number of hydrolase reaction, 2.3fold increase in Km-value for UDP-galactose in hydrolase reaction
W314Y
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29fold decrease in turnover number of galactosyltransferase reaction, 1.67fold increase in Km-value for UDP-galactose in galactosyltransferase reaction, 2fold decrease in Km-value for lactose, 1.1fold increase in turnover number of hydrolase reaction, 1.4fold increase in Km-value for UDP-galactose in hydrolase reaction. Mutation does not affect the overall structure of the enzyme or its interactions with ligands
W356T
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12.3fold decrease in turnover number of galactosyltransferase reaction, 2fold increase in Km-value for UDP-galactose in galactosyltransferase reaction, 6.3fold increase in Km-value for lactose, 14.5fold decrease in turnover number of hydrolase reaction, 2.5fold increase in Km-value for UDP-galactose in hydrolase reaction
N375V
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exchange and truncation of terminal V376, 70% loss of activity
D116A
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the mutant remains active
D118A
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the mutant remains active
D28A
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the mutant remains active
D30A
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the mutant remains active
D316E
mutant show modest reduction in kcat and Km for lactose. Strucutural studies with mutant D316E show that the negative charge is crucial for catalytic activity and needed for its interaction with Arg202 for an active site structure that facilitates the binding of UDP-gal in a catalytically competent conformation
D316E
site-directed mutagenesis, a catalytic domain mutant, crystal structure determination with bound UDP-Gal and Mn2+
D316N
mutant is inactive. Strucutural studies with mutant D316N show that the negative charge is crucial for catalytic activity and needed for its interaction with Arg202 for an active site structure that facilitates the binding of UDP-gal in a catalytically competent conformation
D316N
site-directed mutagenesis, a catalytic domain mutant, crystal structure determination with bound UDP-Gal and Mn2+
E317Q
turnover number for transferase reaction is approximately 2400times lower than that of the wild-type enzyme 120fold reduction in hydrolysis of UDP-galactose. Km-value for UDP-galactose is unchanged
E317Q
crystal structure of the complex of mutant E317Q with UDP-galactose exhibiting a bent configuration stabilized by interactions of the galactose with multiple residues in the enzyme including those in a highly conserved region (His315 to Ser318) is shown
E317Q
site-directed mutagenesis, a catalytic domain mutant, crystal structure determination with bound UDP-Gal and Mn2+
additional information
attaching beta p-nitrophenyl to galactose converts the complex from a poor into a good substrate, the group mimics a monosaccharide like N-acetylglucosamine
additional information
mutants with modified donor substrate specificity
additional information
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mutants with modified donor substrate specificity
additional information
mutated enzymes transfer N-acetylgalactosamine or C2-modified galactose from their UDP derivatives, mutations on the sugar-donor-binding residues (histidine 280, alanine 281, alanine 282) to establish changed specificity, 5-19% of original galactose transferase activity remains, , acceptor affinity is affected by mutation of histidine 280, additional mutation of S347A in all mutants does not affect the enzyme activity and kinetic parameter by itself but resembles the human blood group A and B glycosyltransferases more closely
additional information
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mutated enzymes transfer N-acetylgalactosamine or C2-modified galactose from their UDP derivatives, mutations on the sugar-donor-binding residues (histidine 280, alanine 281, alanine 282) to establish changed specificity, 5-19% of original galactose transferase activity remains, , acceptor affinity is affected by mutation of histidine 280, additional mutation of S347A in all mutants does not affect the enzyme activity and kinetic parameter by itself but resembles the human blood group A and B glycosyltransferases more closely
additional information
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truncation of 3 amino acid residues from the C-terminus, and a frame shift mutation result in a complete loss of activity
additional information
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porcine aortic endothelial cells from control (Gal+/+) and Gal-deficient (Gal-/-) pigs are incubated with human lepirudin anticoagulated whole blood from healthy donors. E-selectin expression is measured by flow cytometry. The C3 inhibitor compstatin and a C5aR antagonist is used to study the role of complement. Gal+/+ porcine aortic endothelial cells incubated with human whole blood show a marked complement C5b-9 dependent up-regulation of E-selectin and secretion of porcine IL-6 and IL-8. Gal-/- cells respond with weak E-selectin and cytokine expression so that the role of complement can not be determined. Human IL-6, IL-8, IFN-gamma, MIP-1alpha, MIP-1beta, eotaxin, and RANTES are detected in the Gal+/+ system, but no responses is seen in the Gal-/- system
additional information
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generation of enzyme knockout mutant animals