The Subunit Structure of Mammalian Fructose Diphosphate Aldolase<sup>*</sup>

Penhoet, Edward E.; Kochman, Marian; Valentine, Raymond C.; Rutter, William J.

doi:10.1021/bi00861a039

Cited by 221 publications

(93 citation statements)

References 37 publications

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“…The tetrameric structure of rabbit muscle aldolase has been well described (Penhoet et al, 1967;Horecker et al, 1972). The stability (Lebherz, 1972) and conservation (Lebherz & Rutter, 1969) of this tetramer was well established, although the role of this structure has remained unknown.…”

Section: Discussionmentioning

confidence: 90%

“…Vertebrate isozymes are thought to operate through a common mechanism involving Schiff base formation between the €-amino group of Lys-229 and the carbonyl carbon of the ketose (Horecker et al, 1972). Aldolase A from muscle exists as an isologous homotetramer, with a subunit molecular mass of 40 kDa (Penhoet et al, 1967). Rabbit aldolase A is well characterized both functionally (Horecker et al, 1972;Berthiaume et al, 1993;Morris & Tolan, 1993) and structurally; a 2.7-A structure has been reported (Sygusch et al, 1987), and it is now resolved to 1.9 A (J.…”

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confidence: 99%

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Subunit interface mutants of rabbit muscle aldolase form active dimers

Beernink

Tolan

1994

Protein Science

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We report the construction of subunit interface mutants of rabbit muscle aldolase A with altered quaternary structure. A mutation has been described that causes nonspherocytic hemolytic anemia and produces a thermolabile aldolase (Kishi H et al., 1987, Proc Natl Acud Sci USA 8436234627). The disease arises from substitution of Gly for Asp-128, a residue at the subunit interface of human aldolase A. To elucidate the role of this residue in the highly homologous rabbit aldolase A, site-directed mutagenesis is used to replace Asp-128 with Gly, Ala, Asn, Gln, or Val. Rabbit aldolase Dl280 purified from Escherichia coli is found to be similar to human D128G by kinetic analysis, CD, and thermal inactivation assays. All of the mutant rabbit aldolases are similar to the wildtype rabbit enzyme in secondary structure and kinetic properties. In contrast, whereas the wild-type enzyme is a tetramer, chemical crosslinking and gel filtration indicate that a new dimeric species exists for the mutants. In sedimentation velocity experiments, the mutant enzymes exist as mixtures of dimer and tetramer at 4 "C. Sedimentation at 20 "C shows that the mutant enzymes are >99.5% dimeric and, in the presence of substrate, that the dimeric species is active. Differential scanning calorimetry demonstrates that T, values of the mutant enzymes are decreased by 12 "C compared to wild-type enzyme. The results indicate that Asp-128 is important for interface stability and suggest that 1 role of the quaternary structure of aldolase is to provide thermostability.

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Section: Discussionmentioning

confidence: 90%

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confidence: 99%

Subunit interface mutants of rabbit muscle aldolase form active dimers

Beernink

Tolan

1994

Protein Science

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“…The subunit structure was studied by cross-linking the purified enzyme with dimethyl suberimidate according to Davies and Stark [I21 with minor modifications using tetrameric aldolase from rabbit muscle as a control [13]. Aldolase and P. furiosus glutamate dehydrogenase were incubated with dimethyl suberimidate in a 1 : 100 and 1 : 5000 ratio, respectively, in 100 mM triethanolamine/HCl, pH 8.5.…”

Section: Molecular Muss Determinationsmentioning

confidence: 99%

Extremely thermostable glutamate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus furiosus

Consalvi

Chiaraluce

Politi

et al. 1991

European Journal of Biochemistry

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The hyperthermophilic archaebacterium Pyrococcus furiosus contains high levels of NAD(P)-dependent glutamate dehydrogenase activity. The enzyme could be involved in the first step of nitrogen metabolism, catalyzing the conversion of 2-oxoglutarate and ammonia to glutamate. The enzyme, purified to homogeneity, is a hexamer of 290 kDa (subunit mass 48 kDa). Isoelectric-focusing analysis of the purified enzyme showed a pl of 4.5. The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate but utilizes both NADH and NADPH as cofactors. The purified enzyme reveals an outstanding thermal stability (the half-life for thermal inactivation at 100°C was 12 h), totally independent of enzyme concentration.P. furiosus glutamate dehydrogenase represents 20% of the total protein; this elevated concentration raises questions about the roles of this enzyme in the metabolism of P..furiosus.Recently we reported the purification of an NAD(P)-dependent glutamate dehydrogenase from the thermophilic archaebacterium Sulfolobus so@ztaricus [l, 21. The enzyme of this microorganism is probably involved in the first step of ammonia assimilation by the following reaction: glutamate + NAD(P)+ + H 2 0 + 2-oxoglutarate + NH,' + NAD(P)H + H + .The glutamate dehydrogenase from S . solfutaricus presents some interesting properties; it has a double coenzyme specificity, is strictly specific for L-glutamate and 2-oxoglutarate and its thermal stability is a function of the enzyme concentration [2]. This latter property is in good agreement with the observed self-associating behaviour of the purified enzyme.Among archaebacterial glutamate dehydrogenases, the only data available for comparison are restricted to the halophilic phenotype [3]. Therefore, it is difficult to extrapolate from our observations on S. solfituricus glutamate dehydrogenase to obtain general rules for thermal adaptation in this class of enzymes and general knowledge regarding nitrogen metabolism in archaebacteria.To provide more information about the enzymological properties of archaebacterial glutamate dehydrogenases and to extend our investigations towards more thermostable forms of this enzyme, we attampted to purify glutamate dehydrogenase from the so-called 'hyperthermophiles'. These remarkable microorganisms, recently discovered, grow optimally at temperatures near 100 "C [4]. They all are archaebacteria and most of them are strict anaerobes and depend on the reduction of elemental sulfur for growth. So far, hyperthermophiles -___ have been classified into three distinct genera, Pyrodiictium, Pyrobaculurn and Pyrococcus. As a source of hyperthermophilic glutamate dehydrogenase we used Pyrococcus furiosus [5], one of the most interesting members of this last genus because of its relatively high cell yield and rapid growth rate. The microorganism grows optimally at 10O'C by a fermentative-type metabolism and is a strict heterotroph which utilizes both simple and complex carbohydrates and produces only H2 and C 0 2 as detectable products. P. furiosus reduces eleme...

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“…The glycolytic enzyme fructose-i, 6-bisphosphate aldolase (aldolase; EC 4.1.2.13) is a tetrameric protein composed of a specific combination of different subunits A (muscle type), B (liver type) and C (brain type) (Penhoet et a!., 1967). These isozymes have the same molecular size (M= 40,000 each), differ in antigenic properties and in net charges, and have been identified and characterized in many mammals (Horecker et a!., 1972;Schapira et a!., 1975;Lebherz and Rutter, 1969).…”

Section: Introductionmentioning

confidence: 99%

“…The genes encoding these subunits appear to be located separately on the chromosomes. Nevertheless, they seem to be closely related structurally: hence they are thought to have originated by gene duplication during evolution (Penhoet et aL, 1967;Benfield et aL, 1979, Lai, 1975.…”

Section: Introductionmentioning

confidence: 99%

Aldolase DNA polymorphism in subterranean mole-rats: genetic differentiation and environmental correlates

et al. 1990

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We analysed the genetic diversity and environmental correlates of the aldolase A and B genes by means of restriction endonucleases (DNA RFLP analysis), in the four chromosomal species (2n =52, 54, 58 and 60) of the actively speciating subterranean mole-rats of the Spalax ehrenbergi superspecies in Israel. The results indicated that: (i) both aldolase genes are highly polymorphic; (ii) Fragment frequencies and fragment profiles display geographical patterns and significant ecological correlates; (iii) discriminant analysis largely succeeded in separating the four chromosomal species on the basis of variation of aldolase RFLPs.

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The Subunit Structure of Mammalian Fructose Diphosphate Aldolase^*

Cited by 221 publications

References 37 publications

Subunit interface mutants of rabbit muscle aldolase form active dimers

Subunit interface mutants of rabbit muscle aldolase form active dimers

Extremely thermostable glutamate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus furiosus

Aldolase DNA polymorphism in subterranean mole-rats: genetic differentiation and environmental correlates

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The Subunit Structure of Mammalian Fructose Diphosphate Aldolase*

Cited by 221 publications

References 37 publications

Subunit interface mutants of rabbit muscle aldolase form active dimers

Subunit interface mutants of rabbit muscle aldolase form active dimers

Extremely thermostable glutamate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus furiosus

Aldolase DNA polymorphism in subterranean mole-rats: genetic differentiation and environmental correlates

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The Subunit Structure of Mammalian Fructose Diphosphate Aldolase^*