Allosteric Regulation of Human Liver Pyruvate Kinase by Peptides that Mimic the Phosphorylated/Dephosphorylated N-Terminus

Glutathione transferases (GSTs) of a novel class, which it is proposed to term Theta, were purified from rat and human liver. Two, named GST 5-5 and GST 12-12, were obtained from the rat, and one, named GST theta, was from the human. Unlike other mammalian GSTs they lack activity towards 1-chloro-2,4-dinitrobenzene and are not retained by GSH affinity matrices. Only GST 5-5 retains full activity during purification, and its activities towards the substrates 1,2-epoxy-3-(p-nitrophenoxy)propane, p-nitrobenzyl chloride, p-nitrophenethyl bromide, cumene hydroperoxide, dichloromethane and DNA hydroperoxide are 185, 86, 67, 42, 11 and 0.03 mumol/min per mg of protein respectively. Earlier preparations of GST 5-5 or GST E were probably a mixture of GST 5-5 and GST 12-12, which was largely inactive, and may also have been contaminated by less than 1% with another GSH peroxidase of far greater activity. Partial analysis of primary structure shows that subunits 5, 12 and theta are related to each other, particularly at the N-terminus, where 25 of 27 residues are identical, but have little relationship to the Alpha, Mu and Pi classes of mammalian GSTs. They do, however, show some relatedness to subunit I of Drosophila melanogaster [Toung, Hsieh & Tu (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 31-35] and the dichloromethane dehalogenase of Methylobacterium DM4 [La Roche & Leisinger (1990) J. Bacteriol, 172, 164-171].

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Molecular Modelling of Human Red Blood Cell Pyruvate Kinase: Structural Implications of a Novel G1091 to A Mutation Causing Severe Nonspherocytic Hemolytic Anemia

Blood ◽

10.1182/blood.v90.12.4987 ◽

1997 ◽

Vol 90 (12) ◽

pp. 4987-4995 ◽

Cited By ~ 20

Author(s):

Wouter W. van Solinge ◽

Rob J. Kraaijenhagen ◽

Gert Rijksen ◽

Richard van Wijk ◽

Bjarne B. Stoffer ◽

...

Keyword(s):

Blood Cell ◽

Pyruvate Kinase ◽

Hemolytic Anemia ◽

Red Blood Cell ◽

Molecular Modelling ◽

Human Liver ◽

The Other ◽

Salt Bridges ◽

Compound Heterozygotes ◽

Allosteric Properties

Abstract We present a novel G1091 to A mutation in the human liver and red blood cell (RBC) pyruvate kinase (PK) gene causing severe hemolytic anemia. In two families, three children were severely PK-deficient compound heterozygotes exhibiting the G1091 to A mutation and a common G1529 to A mutation on the other allele. In one family, the mother, a G1091 to A heterozygote, later had a second baby with a new husband, also a G1091 to A carrier. The baby was homozygous for the G1091 to A mutation and died 6 weeks after birth from severe hemolysis. Both mutant alleles were expressed at the RNA level. The G1091 to A mutation results in the substitution of a conserved glycine by an aspartate in domain A of RBC PK, whereas the G1529 to A mutation leads to the substitution of a conserved arginine residue with glutamine in the C-domain. Molecular modelling of human RBC PK, based on the crystal structure of cat muscle PK, shows that both mutations are located outside the catalytic site at the interface of domains A and C. The mutations are likely to disrupt the critical conformation of the interface by introducing alternative salt bridges. In this way the Gly364 to Asp and Arg510 to Gln substitutions may cause PK deficiency by influencing the allosteric properties of the enzyme.

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