Purification, properties and kinetic analysis of UDP-glucose pyrophosphorylase from bovine mammary tissue

Aksamit, Robert R.; Ebner, Kurt E.

doi:10.1016/0005-2744(72)90203-3

Cited by 8 publications

(5 citation statements)

References 45 publications

(49 reference statements)

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“…In the reverse reaction (UDP-Glc and MgPP, as substrates), the reaction was monitored at 340 nm by addition of phosphoglucomutase (5 Uiml) and its cofactor Glc(1,6)P, (2 pM), Glc6P dehydrogenase ( 5 U/ ml) and NADP' (1 mM). For assays in both directions, MgCI, was added to a total concentration calculated so as to g' rive a free magnesium ion concentration of 1 mM, using the stability constants cited by Aksamit and Ebner (1972) for the Mg complexes of the reactants.…”

Section: Methodsmentioning

confidence: 99%

Sequence Differences between Human Muscle and Liver cDNAs for UDPglucose Pyrophosphorylase and Kinetic Properties of the Recombinant Enzymes Expressed in Escherichia Coli

Duggleby

Huang

et al. 1996

European Journal of Biochemistry

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UDP-Glc pyrophosphorylase (EC 2.7.7.9) catalyses the interconversion of MgUTP plus Glcl P and UDP-Glc plus MgPP,. Complementation of an Escherichia coli strain lacking this activity has allowed isolation of cDNA encoding this enzyme from a human muscle library. Two forms were identified and the nucleotide sequence of each was determined; they were found to differ only in the 5' region and we suggest that these arise from the use of a different first exon in the two transcripts. These nucleotide sequences are different from that of the cDNA which was isolated previously from a human liver library Lett. 329,[153][154][155][156][157][158].1 and it is proposed that these liver and muscle forms are derived from different genes. The cDNA for muscle form I, muscle form 11, the liver form, and the liver form fused to part of the lacZ gene were expressed in Escherichiu coli and the kinetic properties of each enzyme were characterised. Muscle form I and the LacZAiver fusion enzyme exhibit Michaelis-Menten kinetics towards all substrates while muscle form TI has a sigmoidal dependence of rate upon the concentration of MgPP,. The liver form shows Michaelis-Menten kinetics towards MgUTP. For the remaining three substrates, complex kinetics were observed involving a combination of sigmoidicity at low substrate concentration and partial inhibition at high substrate concentration.

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Section: Methodsmentioning

confidence: 99%

Sequence Differences between Human Muscle and Liver cDNAs for UDPglucose Pyrophosphorylase and Kinetic Properties of the Recombinant Enzymes Expressed in Escherichia Coli

Duggleby

Huang

et al. 1996

European Journal of Biochemistry

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“…UGPase (UDP-glucose pyrophosphorylase) (EC 2.7.7.9) which is ubiquitously distributed in micro-organisms, plants and animals, plays very important roles in carbohydrate metabolism. UGPase can reversibly catalyse the formation of UDP-glucose and pyrophosphate from UTP and glucose 1-phosphate in the presence of Mg 2 + [1]. UDP-glucose, the active form of glucose, plays very important roles in saccharide metabolism.…”

Section: Introductionmentioning

confidence: 99%

“…The atomic co-ordinates and structural factors of human UDP-glucose pyrophosphorylase have been deposited in the RCSB PDB under accession code 3R2W. 1 To whom correspondence should be addressed (email xiaofengz@pku.edu.cn).…”

Section: Introductionmentioning

confidence: 99%

The crystal structure of human UDP-glucose pyrophosphorylase reveals a latch effect that influences enzymatic activity

Zheng

2012

Biochemical Journal

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UGPase (UDP-glucose pyrophosphorylase) is highly conserved among eukaryotes. UGPase reversibly catalyses the formation of UDP-glucose and is critical in carbohydrate metabolism. Previous studies have mainly focused on the UGPases from plants, fungi and parasites, and indicate that the regulatory mechanisms responsible for the enzyme activity vary among different organisms. In the present study, the crystal structure of hUGPase (human UGPase) was determined and shown to form octamers through end-to-end and side-by-side interactions. The observed latch loop in hUGPase differs distinctly from yUGPase (yeast UGPase), which could explain why hUGPase and yUGPase possess different enzymatic activities. Mutagenesis studies showed that both dissociation of octamers and mutations of the latch loop can significantly affect the UGPase activity. Moreover, this latch effect is also evolutionarily meaningful in UGPase from different species.

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“…In the reverse reaction, (UDP-Glc and MgPP, as substrates) the reaction was monitored at 340 nm by addition of phosphoglucomutase (5 U/ml) and its cofactor Glc(l,6)P2 (2 pM), Glc6P dehydrogenase (5 U/ml) and NADP' (1 mM). For assays in both directions, MgCI, was added to a total concentration calculated so as to give a free magnesium ion concentration of 1 mM, using the stability constants cited by Aksamit and Ebner (1972) for the Mg complcxes of the reactants.…”

Section: Methodsmentioning

confidence: 99%

The Importance of Conserved Residues in Human Liver UDPglucose Pyrophosphorylase

Chang

Peng

et al. 1996

European Journal of Biochemistry

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Comparison of the amino acid sequences of five eukaryotic UDPglucose pyrophosphorylases has identified a number of conserved residues that may be important for substrate binding or catalysis. Using the cloned cDNA for the human liver enzyme, we have investigated the role of several of these residues by site-directed mutagenesis. Changing the single conserved cysteine (residue 123) to serine resulted in an active enzyme, as did mutating the single conserved histidine (residue 266) to arginine. The two conserved tryptophans were each altered to serine; W218S is active while W333S is not. In the latter case, the enzyme does not appear to fold correctly, and a similar result was obtained by mutation to lysine at one (residue 391) of the four conserved arginines. The other three arginines are not essential, as judged by the observation that R389H, R422Q and R445H are all active. The kinetic properties of each active mutant were investigated and in most cases were found to be similar to those of wild-type. The most dramatic change is a sevenfold increase in the K,,, for magnesium pyrophosphate with C123S. Overall, none of these conserved residues appears to be essential for activity, although such a role cannot be ruled out for W333 and R391 where mutation resulted in defective folding.

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Purification, properties and kinetic analysis of UDP-glucose pyrophosphorylase from bovine mammary tissue

Cited by 8 publications

References 45 publications

Sequence Differences between Human Muscle and Liver cDNAs for UDPglucose Pyrophosphorylase and Kinetic Properties of the Recombinant Enzymes Expressed in Escherichia Coli

Sequence Differences between Human Muscle and Liver cDNAs for UDPglucose Pyrophosphorylase and Kinetic Properties of the Recombinant Enzymes Expressed in Escherichia Coli

The crystal structure of human UDP-glucose pyrophosphorylase reveals a latch effect that influences enzymatic activity

The Importance of Conserved Residues in Human Liver UDPglucose Pyrophosphorylase

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