Crystal structure of human muscle aldolase complexed with fructose 1,6-bisphosphate: mechanistic implications.
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Fructose 1,6-bisphosphate aldolase catalyzes the reversible cleavage of fructose 1,6-bisphosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceraldehyde 3-phosphate or glyceraldehyde, respectively. Catalysis involves the formation of a Schiff's base intermediate formed at the epsilon-amino group of Lys229. The existing apo-enzyme structure was refined using the crystallographic free-R-factor and maximum likelihood methods that have been shown to give improved structural results that are less subject to model bias. Crystals were also soaked with the natural substrate (fructose 1,6-bisphosphate), and the crystal structure of this complex has been determined to 2.8 A. The apo structure differs from the previous Brookhaven-deposited structure (1ald) in the flexible C-terminal region. This is also the region where the native and complex structures exhibit differences. The conformational changes between native and complex structure are not large, but the observed complex does not involve the full formation of the Schiff's base intermediate, and suggests a preliminary hydrogen-bonded Michaelis complex before the formation of the covalent complex. (+info)
Kinetic properties and tissular distribution of mammalian phosphomannomutase isozymes.
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Human tissues contain two types of phosphomannomutase, PMM1 and PMM2. Mutations in the PMM2 gene are responsible for the most common form of carbohydrate-deficient glycoprotein syndrome [Matthijs, Schollen, Pardon, Veiga-da-Cunha, Jaeken, Cassiman and Van Schaftingen (1997) Nat. Genet. 19, 88-92]. The protein encoded by this gene has now been produced in Escherichia coli and purified to homogeneity, and its properties have been compared with those of recombinant human PMM1. PMM2 converts mannose 1-phosphate into mannose 6-phosphate about 20 times more rapidly than glucose 1-phosphate to glucose 6-phosphate, whereas PMM1 displays identical Vmax values with both substrates. The Ka values for both mannose 1,6-bisphosphate and glucose 1,6-bisphosphate are significantly lower in the case of PMM2 than in the case of PMM1. Like PMM1, PMM2 forms a phosphoenzyme with the chemical characteristics of an acyl-phosphate. PMM1 and PMM2 hydrolyse different hexose bisphosphates (glucose 1,6-bisphosphate, mannose 1,6-bisphosphate, fructose 1,6-bisphosphate) at maximal rates of approximately 3.5 and 0.3% of their PMM activity, respectively. Fructose 1,6-bisphosphate does not activate PMM2 but causes a time-dependent stimulation of PMM1 due to the progressive formation of mannose 1,6-bisphosphate from fructose 1,6-bisphosphate and mannose 1-phosphate. Experiments with specific antibodies, kinetic studies and Northern blots indicated that PMM2 is the only detectable isozyme in most rat tissues except brain and lung, where PMM1 accounts for about 66 and 13% of the total activities, respectively. (+info)
Expression and regulation of 6-phosphofructo-2-kinase/fructose- 2,6-bisphosphatase isozymes in white adipose tissue.
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The aim of this work was to identify the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) isozyme(s) present in white adipose tissue. Ion-exchange chromatography of PFK-2 from rat epididymal fat pads yielded an elution pattern compatible with the presence of both the L (liver) and M (muscle) isozymes. This was consistent with a study of the phosphorylation of the purified adipose tissue enzyme by cAMP-dependent protein kinase, by specific labelling of the preparation with [2-32P]fructose 2,6-bisphosphate and by reaction with antibodies. Characterization of the PFK-2/FBPase-2 mRNAs showed that mature adipocytes express the mRNA that codes for the L isozyme and the two mRNAs that code for the M isozyme. Preadipocytes expressed mRNA that codes for the M isozyme. Incubation of rat epididymal fat pads with adrenaline stimulated glycolysis but decreased fructose 2,6-bisphosphate concentrations without significant inactivation of PFK-2. These results support previous findings showing that fructose 2,6-bisphosphate is not involved in the adrenaline-induced stimulation of glycolysis in white adipose tissue. (+info)
Chloroplast class I and class II aldolases are bifunctional for fructose-1,6-biphosphate and sedoheptulose-1,7-biphosphate cleavage in the Calvin cycle.
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Class I and class II aldolases are products of two evolutionary non-related gene families. The cytosol and chloroplast enzymes of higher plants are of the class I type, the latter being bifunctional for fructose-1,6- and sedoheptulose-1,7-P2 in the Calvin cycle. Recently, class II aldolases were detected for the cytosol and chloroplasts of the lower alga Cyanophora paradoxa. The respective chloroplast enzyme has been shown here to be also bifunctional for fructose-1,6- and sedoheptulose-1,7-P2. Kinetics, also including fructose-1-P, were determined for all these enzymes. Apparently, aldolases are multifunctional enzymes, irrespective of their class I or class II type. (+info)
Apoptosis induced by growth factor withdrawal in fibroblasts overproducing fructose 2,6-bisphosphate.
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Fructose 2,6-bisphosphate is a potent endogenous stimulator of glycolysis. A high aerobic glycolytic rate often correlates with increased cell proliferation. To investigate this relationship, we have produced clonal cell lines of Rat-1 fibroblasts that stably express transgenes coding for 6-phosphofructo-2-kinase, which catalyzes the synthesis of fructose 2,6-bisphosphate, or for fructose 2,6-bisphosphatase, which catalyzes its degradation. While serum deprivation in culture reduced the growth rate of control cells, it caused apoptosis in cells overproducing fructose 2,6-bisphosphate. Apoptosis was inhibited by 5-amino-4-imidazolecarboxamide riboside, suggesting that 5'-AMP-activated protein kinase interferes with this phenomenon. (+info)
Mutational analysis of the role of HPr in Listeria monocytogenes.
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The regulatory role of HPr, a protein of the phosphotransferase system (PTS), was investigated in Listeria monocytogenes. By constructing mutations in the conserved histidine 15 and serine 46 residues of HPr, we were able to examine how HPr regulates PTS activity. The results indicated that histidine 15 was phosphorylated in a phosphoenolpyruvate (PEP)-dependent manner and was essential for PTS activity. Serine 46 was phosphorylated in an ATP-dependent manner by a membrane-associated kinase. ATP-dependent phosphorylation of serine 46 was significantly enhanced in the presence of fructose 1,6-diphosphate and resulted in a reduction of PTS activity. The presence of a charge at position 15 did not inhibit ATP-dependent phosphorylation of serine 46, a finding unique to gram-positive PEP-dependent PTSs studied to this point. Finally, HPr phosphorylated at serine 46 does not appear to possess self-phosphatase activity, suggesting a specific phosphatase protein may be essential for the recycling of HPr to its active form. (+info)
Alteration of substrate specificity by a naturally-occurring aldolase B mutation (Ala337-->Val) in fructose intolerance.
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A molecular analysis of human aldolase B genes in two newborn infants and a 4-year-old child with hereditary fructose intolerance, the offspring of a consanguineous union, has identified the novel mutation Ala337-->Val in homozygous form. This mutation was also detected independently in two other affected individuals who were compound heterozygotes for the prevalent aldolase B allele, Ala149-->Pro, indicating that the mutation causes aldolase B deficiency. To test for the effect of the mutation, catalytically active wild-type human aldolase B and the Val337 variant enzyme were expressed in Escherichia coli. The specific activities of the wild-type recombinant enzyme were 4.8 units/mg and 4.5 units/mg towards fructose 1,6-bisphosphate (FBP) and fructose 1-phosphate (F-1-P) as substrates with Michaelis constants of 4 microM and 2.4 mM respectively. The specific activities of purified tetrameric Val337 aldolase B, which affects an invariant residue in the C-terminal region, were 4.2 units/mg and 2.6 units/mg towards FBP and F-1-P as substrates respectively; the corresponding Michaelis constants were 22 microM and 24 mM. The FBP-to-F-1-P substrate activity ratios were 0.98 and 1.63 for wild-type and Val337 variant enzymes respectively. The Val337 mutant aldolase had an increased susceptibility to proteolytic cleavage in E. coli and rapidly lost activity on storage. Comparative CD determinations showed that the Val337 protein had a distinct thermal denaturation profile with markedly decreased enthalpy, indicating that the mutant protein is partly unfolded. The undegraded mutant had preferentially decreased affinity and activity towards its specific F-1-P substrate and maintained appreciable activity towards FBP. In contrast, fluorescence studies of the mutant showed an increased binding affinity for products of the aldolase reaction, indicating a role for the C-terminus in mediating product release. These findings in a rare but widespread naturally occurring mutant implicate the C-terminus in the activity of human aldolase B towards its specific substrates and demonstrate its role in maintaining the overall stability of the enzyme tetramer. (+info)
Role of quinolinate phosphoribosyl transferase in degradation of phthalate by Burkholderia cepacia DBO1.
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Two distinct regions of DNA encode the enzymes needed for phthalate degradation by Burkholderia cepacia DBO1. A gene coding for an enzyme (quinolinate phosphoribosyl transferase) involved in the biosynthesis of NAD+ was identified between these two regions by sequence analysis and functional assays. Southern hybridization experiments indicate that DBO1 and other phthalate-degrading B. cepacia strains have two dissimilar genes for this enzyme, while non-phthalate-degrading B. cepacia strains have only a single gene. The sequenced gene was labeled ophE, due to the fact that it is specifically induced by phthalate as shown by lacZ gene fusions. Insertional knockout mutants lacking ophE grow noticeably slower on phthalate while exhibiting normal rates of growth on other substrates. The fact that elevated levels of quinolinate phosphoribosyl transferase enhance growth on phthalate stems from the structural similarities between phthalate and quinolinate: phthalate is a competitive inhibitor of this enzyme and the phthalate catabolic pathway cometabolizes quinolinate. The recruitment of this gene for growth on phthalate thus gives B. cepacia an advantage over other phthalate-degrading bacteria in the environment. (+info)