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PubMed1993

Cobalamin metabolism in cultured human chorionic villus cells.

Cobalamin (Cbl, vitamin B12) metabolism was analyzed in cultures of human chorionic villus (CV) cells obtained at 9-10 weeks of gestation. CV cells were shown to synthesize transcobalamin II (TCII) and to possess a high affinity receptor for that molecule. The cells bound and internalized radioactive cyanocobalamin (CN[57Co]Cbl) complexed to TCII. This internalized CN[57Co]Cbl was found to be converted to both methylCbl and adenosylCbl, the two intracellular coenzyme forms of Cbl, and bound to the two known intracellular Cbl requiring enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase. Both enzyme systems were found to be functional in the intact cell by demonstrating the incorporation of the radioactive label from both [14C]CH3-tetrahydrofolate and [14C]propionate into acid insoluble products. MS activity was also detected in lysed cell material. CV cells were shown not to be auxotrophic for methionine since they were able to utilize homocysteine in place of methionine for cell division. Since CV cells are capable of performing many of the complex events associated with Cbl metabolism, it may be possible to use these cells to diagnose genetic defects of Cbl metabolism.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1998

Exploring sequence constraints on an interhelical turn using in vivo selection for catalytic activity.

The role of interhelical turns in determining protein structure has been investigated previously in relatively simple four-helix-bundle proteins using combinatorial mutagenesis coupled with screening for functional variants. To assess the tolerance to sequence substitution of a short, interhelical turn in a larger, more complicated protein, we have exploited a more sensitive in vivo selection for catalytic activity. Randomization of three solvent-exposed turn residues in Escherichia coli chorismate mutase (Ala65, His66, and His67), followed by selection, indicated that >63% of tripeptides, including some with significantly altered backbone conformations, can functionally replace the native sequence. The increased sensitivity of the catalytic assay allowed optimal sequences to be distinguished from less appropriate ones, revealing a statistically significant preference for hydrophilic residues in solvent-exposed positions. It also enabled investigation of the extent to which either secondary structure or tertiary interactions influence substitution patterns. Randomization of an alpha-helical residue (Lys64), together with the adjacent solvent-exposed tripeptide, Ala65-His66-His67, showed that the secondary structure at position 64 does not limit the range of side chains allowed at this site. In contrast, randomization of a buried turn residue (Leu68), together with the same tripeptide, revealed an extremely strict requirement for hydrophobic aliphatic amino acids at this position. The strong constraint imposed by the tertiary interaction, in contrast to the weak influence of secondary structure, has important implications for protein design.

Catalysis↗

PubMed1998

Probing enzyme quaternary structure by combinatorial mutagenesis and selection.

Genetic selection provides an effective way to obtain active catalysts from a diverse population of protein variants. We have used this tool to investigate the role of loop sequences in determining the quaternary structure of a domain-swapped enzyme. By inserting random loops of four to seven residues into a dimeric chorismate mutase and selecting for functional variants by genetic complementation, we have obtained and characterized both monomeric and hexameric enzymes that retain considerable catalytic activity. The low percentage of active proteins recovered from these selection experiments indicates that relatively few loop sequences permit a change in quaternary structure without affecting active site structure. The results of our experiments suggest further that protein stability can be an important driving force in the evolution of oligomeric proteins.

Chorismate Mutase↗

PubMed2001

Conserved core structure and active site residues in alkaline phosphatase superfamily enzymes.

Cofactor-independent phosphoglycerate mutase (iPGM) has been previously identified as a member of the alkaline phosphatase (AlkP) superfamily of enzymes, based on the conservation of the predicted metal-binding residues. Structural alignment of iPGM with AlkP and cerebroside sulfatase confirmed that all these enzymes have a common core structure and revealed similarly located conserved Ser (in iPGM and AlkP) or Cys (in sulfatases) residues in their active sites. In AlkP, this Ser residue is phosphorylated during catalysis, whereas in sulfatases the active site Cys residues are modified to formylglycine and sulfatated. Similarly located Thr residue forms a phosphoenzyme intermediate in one more enzyme of the AlkP superfamily, alkaline phosphodiesterase/nucleotide pyrophosphatase PC-1 (autotaxin). Using structure-based sequence alignment, we identified homologous Ser, Thr, or Cys residues in other enzymes of the AlkP superfamily, such as phosphopentomutase, phosphoglycerol transferase, phosphonoacetate hydrolase, and GPI-anchoring enzymes (glycosylphosphatidylinositol phosphoethanolamine transferases) MCD4, GPI7, and GPI13. We predict that catalytical cycles of all the enzymes of AlkP superfamily include phosphoenzyme (or sulfoenzyme) intermediates.

Alkaline Phosphatase↗

PubMed1993

YMC1, a yeast gene encoding a new putative mitochondrial carrier protein.

We have cloned and sequenced a Saccharomyces cerevisiae gene coding for a protein with significant similarities to the mitochondrial carrier family. The gene we termed YMC1 (yeast mitochondrial carrier) is located on chromosome XVI, closely downstream of ARO7 encoding chorismate mutase.

Amino Acid Sequence↗

PubMed1997

A study on the complexes between human erythrocyte enzymes participating in the conversions of 1,3-diphosphoglycerate.

The ability of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzing the reaction of 1,3-diphosphoglycerate synthesis in human erythrocytes to form complexes with enzymes which use this metabolite as substrate (3-phosphoglycerate kinase (3-PGK) or 2,3-diphosphoglycerate mutase (2,3-DPGM)) was studied. It was found that highly active 2,3-DPGM can be extracted from human erythrocyte hemolysates in a complex with GAPDH adsorbed on Sepharose-bound anti-GAPDH antibodies at pH 6.5, the molar ratio being one 2,3-GPGM subunit per subunit of GAPDH. No complexation was, however, detected at pH 8.0. The opposite was true for the interaction between GAPDH and 3-PGK, which could be observed at pH 8.0. In experiments carried out at pH 7.4, both GAPDH x 2,3-DPGM and GAPGH x 3-PGK complexes were detected. The Kd values of the complexes determined with purified enzyme preparations were in the range 2.40-2.48 microM for both the GAPDH x 2,3-DPGM and GAPGH x 3-PGK enzyme pairs, when titrations of GAPDH covalently bound to CNBr-activated Sepharose were performed by the soluble 2,3-DPGM or 3-PGK. If, however, GAPDH adsorbed on the specific antibodies covalently bound to Sepharose was used in the titration experiments, the Kd for the GAPDH x 2,3-DPGM complex was found to be 0.54 microM, and the Kd for the GAPDH x 3-PGK complex was 0.49 microM. The concentration of 2,3-diphosphoglycerate determined after 1 h of incubation of erythrocytes in the presence of glucose was found to increase 1.5-fold if the incubation was carried out at pH 6.5, but did not change upon incubation at pH 8.0. On the other hand, the concentration of 3-phosphoglycerate after incubation at pH 8.0 was twice as large as that found after incubation at pH 6.5. The results are interpreted on the hypothesis that specific protein-protein interactions between GAPDH and 2,3-DPGM or between GAPDH and 3-PGK may play a role in determining the fate of 1,3-diphosphoglycerate produced in the GAPDH-catalyzed reaction.

2,3-Diphosphoglycerate↗

PubMed1997

Enhanced levels of biochemical markers for cobalamin deficiency in totally gastrectomized rats: uncoupling of the enhancement from the severity of spongy vacuolation in spinal cord.

The totally gastrectomized (TGX) rat is a new experimental model for studying the pathogenesis of cobalamin (Cbl)-deficient myelopathy, i.e., subacute combined degeneration, total gastrectomy (TG) serving as a surgical paradigm of human pernicious anemia. We determined the serum levels of some biochemical indicators of Cbl deficiency in TGX rats at 2 to 10 months after TG. Methylmalonic acid (MMA) rose within 2 months and progressively increased thereafter until the end of the investigation period. 2-Methylcitric acid (MCA) rose significantly by 6 months and showed a further increment 4 months later. Homocysteine was only clearly elevated much later than the serum MMA, i.e., 10 months after the operation. The concentrations of MMA, MCA, and cystathionine were increased in kidney, liver, and spinal cord (SC) of TGX rats at 10 months. Chronic treatment of TGX rats with Cbl greatly decreased the serum levels of all the metabolic indicators of Cbl deficiency. Chronic peroral administration of the antibiotic lincomycin to TGX rats in an attempt to suppress the enteric flora markedly decreased serum MMA levels. Only Cbl, however, given either for the first 2 months after TG or for the third and fourth postoperative months (i.e., after SC abnormalities had already appeared) significantly decreased the severity of spongy vacuolation in SC white matter, although not completely preventing or repairing the neuropathological damage. Therefore, neither the early impairment in TGX rats of the Cbl-dependent methylmalonyl-coenzyme A mutase reaction nor the more delayed impairment of the Cbl-dependent methionine synthase step, as reflected by changes in serum metabolite levels, seems to be causally related to the TG-induced spongy vacuolation in SC white matter.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1998

Fine mapping of the autosomal recessive polycystic kidney disease locus (PKHD1) and the genes MUT, RDS, CSNK2 beta, and GSTA1 at 6p21.1-p12.

A total of 33 polymorphic markers were analyzed to generate a high-resolution genetic linkage map of the locus PKHD1 (polycystic kidney and hepatic disease 1) for the autosomal recessive polycystic kidney disease (ARPKD), using a combination of recombination mapping and linkage analysis in 164 families. Recombinants narrowed the PKHD1 region from 3.8 cM to a 1-cM interval flanked by the markers D6S1024 and D6S1714. Linkage disequilibrium analysis in 13 Finnish ARPKD families identified two different highly conserved haplotypes with four distal flanking markers, suggesting the existence of at least two major mutations of Finnish origin. The genes MUT (methylmalonyl coenzyme A-mutase), RDS (retinal degeneration, slow), CSNK2 beta (casein kinase II, beta subunit), and GSTA1 (glutathione S-transferase alpha, type 1) were excluded as PKHD1 genes using both established and novel intragenic polymorphisms in families with key recombinants. These genetic data, combined with our YAC-based physical map of the 6p21-p12 region, will facilitate efforts to positionally clone the PKHD1 gene.

Casein Kinase II↗

PubMed1993

The pheA/tyrA/aroF region from Erwinia herbicola: an emerging comparative basis for analysis of gene organization and regulation in enteric bacteria.

Extensive knowledge exists in Escherichia coli about the contiguous pheA and aroF-tyrA operons which have opposite transcription orientations and are separated by a bidirectional transcription terminator. The corresponding structural genes and individual components of the terminator and attenuator from Erwinia herbicola have been analyzed from an evolutionary vantage point. A 7.5-kb DNA fragment from E. herbicola carrying the linked pheA, tyrA, and aroF genes was cloned by functional complementation of E. coli auxotrophic requirements. A 3,433-bp segment of DNA consisting of more than half of aroF, all of tyrA, and the entire phenylalanine operon (promoter, leader region encoding the leader peptide and containing the phe attenuator, and pheA) was sequenced. A bidirectional transcription terminator was positioned between the divergently transcribed pheA and tyrA. The adjacent aroF and tyrA genes share a common transcription orientation, consistent with their probable coexistence within an operon. However, tyrA can be expressed efficiently from an internal promoter which appears to lie within the 3' portion of aroF. The gene order is pheA tyrA aroF in E. herbicola, with the same tail-to-tail arrangement of transcription known to exist in E. coli. The pheL coding region of the phe operon was dominated by phenylalanine codons, seven of the 15 amino acid residues of the leader peptide being L-phenylalanine. The E. herbicola pheA and tyrA genes were 1,161 bp and 1,119 bp in length, respectively, and corresponded to deduced gene products having subunit molecular weights of 43,182 and 41,847. The deduced amino acid sequences of PheA and TyrA were homologous at their N-termini, consistent with a common evolutionary origin of the chorismate mutase domains present at the amino terminus of both PheA and TyrA. A detailed comparison of the E. coli and E. herbicola sequences was made. The pheA, tyrA, and aroF genes of E. herbicola exhibited high overall identity with the counterpart E. coli genes. Within the leader region of the phe operon, the leader peptide coding region was highly conserved. Although the 1:2 and 2':3' stems defining the pause structure and the antiterminator, respectively, were also highly conserved, RNA segment 4 of the attenuator terminator exhibited considerable divergence, as did the distal portion of the attenuator region. Within the span of attenuator region encoding the three stem-loop structures of mRNA secondary configuration, hot spots of base-residue divergence were localized to looped-out regions. No changes occurred which would simultaneously disrupt alternative pairing relationships of secondary configuration. The bidirectional terminator between pheA and tyrA has diverged very substantially.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Deoxy-7-Phosphoheptulonate Synthase↗

PubMed1993

Phenylalanine production by metabolically engineered Corynebacterium glutamicum with the pheA gene of Escherichia coli.

The bifunctional enzyme chorismate mutase (CM)-prephenate dehydratase (PD), which is encoded by the pheA gene of Escherichia coli, catalyses the two consecutive key steps in phenylalanine biosynthesis. To utilize the enzyme for metabolic engineering of phenylalanine-producing Corynebacterium glutamicum KY10694, the intact gene was cloned on a multicopy vector to yield pEA11.C. glutamicum cells transformed with pEA11 exhibited a more than tenfold increase in CM and PD activities relative to the host cells. Moreover, the level of pheA expression was further elevated a fewfold when cells were starved of phenylalanine, suggesting that the attenuation regulation of pheA expression functions in heterogeneous C. glutamicum. Plasmid pEA11 encoding the wild-type enzyme was mutated to yield pEA22, which specified CM-PD exhibiting almost complete resistance to end-product inhibition. When pEA22 was introduced into KY10694, both the activities of CM and PD were highly maintained throughout the cultivation, thus leading to a 35% increased production (23 g/l) of phenylalanine.

Biotechnology↗

PubMed1991

Increase of 2,3-bisphosphoglycerate synthase/phosphatase during maturation of reticulocytes with high 2,3-bisphosphoglycerate content.

In the rabbit and in the rat, which possess erythrocytes with high concentration of 2,3-bisphosphoglycerate, the 2,3-bisphosphoglycerate synthase activity increases more than two fold during reticulocyte maturation. Isolation of the enzymes with 2,3-bisphosphoglycerate synthase activity present in extracts of reticulocytes and mature erythrocytes by ion exchange fast liquid chromatography shows that the increase in the synthase activity is due to the accumulation of the bifunctional enzyme 2,3-bisphosphoglycerate synthase/phosphatase (EC2.7.5.4/EC3.1.3.13) which represents more than 80% of the synthase activity of the cell extracts. During reticulocyte maturation phosphoglycerate mutase (EC 5.4.2.1), which makes a small contribution to the 2,3-bisphosphoglycerate synthase activity in the erythroid cells, decreases in the rabbit and remains constant in the rat.

2,3-Diphosphoglycerate↗

PubMed1990

A GCN4 protein recognition element is not sufficient for GCN4-dependent regulation of transcription in the ARO7 promoter of Saccharomyces cerevisiae.

The gene ARO7 encodes the monofunctional enzyme chorismate mutase, a branch point enzyme in the aromatic amino acid biosynthetic pathway in Saccharomyces cerevisiae. We investigated the transcription of the ARO7 gene. Three 5' ends at positions -36, -56 and -73 and the 3' end of the transcripts 146 bp downstream of the translational stop codon were mapped. As in the promoters of other aromatic amino acid biosynthetic genes, a recognition element for the GCN4 transcriptional activator of amino acid biosynthesis is located 425 base pairs (bp) upstream of the first transcriptional start point. This element binds GCN4 specifically in vitro. Northern analysis and determination of the specific enzyme activity reveals however, that the element is not sufficient to mediate transcriptional regulation by GCN4 in vivo. We thus suggest that in addition to a consensus sequence capable of binding the GCN4 protein other factors like, for example, chromatin structure, determine whether a recognition site for a transcription factor functions as an upstream activation sequence.

Blotting, Northern↗

PubMed1994

Open reading frames in the antisense strands of genes coding for glycolytic enzymes in Saccharomyces cerevisiae.

Open reading frames longer than 300 bases were observed in the antisense strands of the genes coding for the glycolytic enzymes phosphoglucose isomerase, phosphoglycerate mutase, pyruvate kinase and alcohol dehydrogenase I. The open reading frames on both strands are in codon register. It has been suggested that proteins coded in codon register by complementary DNA strands can bind to each other. Consequently, it was interesting to investigate whether the open reading frames in the antisense strands of glycolytic enzyme genes are functional. We used oligonucleotide-directed mutagenesis of the PGI1 phosphoglucose isomerase gene to introduce pairs of closely spaced base substitutions that resulted in stop codons in one strand and only silent replacements in the other. Introduction of the two stop codons into the PGI1 sense strand caused the same physiological defects as already observed for pgil deletion mutants. No detectable effects were caused by the two stop codons in the antisense strand. A deletion that removed a section from -31 bp to +109 bp of the PGI1 gene but left 83 bases of the 3' region beyond the antisense open reading frame had the same phenotype as a deletion removing both reading frames. A similar pair of deletions of the PYK1 gene and its antisense reading frame showed identical defects. Our own Northern experiments and those reported by other authors using double-stranded probes detected only one transcript for each gene. These observations indicate that the antisense reading frames are not functional. On the other hand, evidence is provided to show that the rather long reading frames in the antisense strands of these glycolytic enzyme genes could arise from the strongly selective codon usage in highly expressed yeast genes, which reduces the frequency of stop codons in the antisense strand.

Base Sequence↗

PubMed1975

Enzymatic analysis of the pathways of glucose catabolism and gluconeogenesis in Pseudomonas citronellolis.

Extracts of Pseudomonas citronellolis cells grown on glucose or gluconate possessed all the enzymes of the Entner-Doudoroff pathway. Gluconokinase and either or both 6-phosphogluconate dehydratase and KDPG aldolase were induced by growth on these substrates. Glucose and gluconate dehydrogenases and 6-phosphofructokinase were not detected. Thus catabolism of glucose proceeds via an inducible Entner-Doudoroff pathway. Metabolism of glyceraldehyde 3-phosphate apparently proceeded via glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase. These same enzymes plus triose phosphate isomerase were present in lactate-grown cells indicating that synthesis of triose phosphates from gluconeogenic substrates also occurs via this pathway. Extracts of lactate grown-cells possessed fructose diphosphatase and phosphohexoisomerase but apparently lacked fructose diphosphate aldolase thus indicating either the presence of an aldolase with unusual properties or requirements or an alternative pathway for the conversion of triose phosphate to fructose disphosphate. Cells contained two species of glyceraldehyde 3-phosphate dehydrogenase, one an NAD-dependent enzyme which predominated when the organism was grown on glycolytic substrates and the other, an NADP-dependent enzyme which predominated when the organism was grown on gluconeogenic substrates.

Fructose-Bisphosphatase↗

PubMed1978

Methylmalonic acidemia.

A patient presenting with severe metabolic acidosis accompanied by hyperglycinemia, hyperuricemia, hypoglycemia and hypertammonemia is described. Metabolic acidosis was found to be due to accumulated methylmalonic acid and did not respond to vitamin B12 administration. The patient showed favorable growth and development when kept on a low isoleucine, methionine, threonine and valine diet. In vitro studies using a lymphoid cell line derived from the patient showed a deficiency of methylmalonyl-CoA carbonyl-mutase.

Acidosis↗

PubMed1978

High pyruvate kinase activity causes low concentration of 2,3-diphosphoglycerate in fetal rabbit red cells.

1. The high oxygen affinity of fetal blood in rabbits is due to a very low concentration of 2,3-diphosphoglycerate (2,3-DPG) in the red cells. In order to gather informations on the factors responsible for this characteristic we have studied synthesis and break-down of 2,3-DPG in fetal and adult rabbit red cells in vitro and examined possible regulative pathways which may lead to the low 2,3-DPG concentration in vivo. 2. Under conditions where 2,3-DPG and 3-phosphoglycerate (3-PGA) accumulate in adult erythrocytes, i.e. in a solution containing inosine, pyruvate and inorganic phosphate, the amount of 2,3-DPG synthetized in fetal red cells was only 40% of the adult value and 3-PGA was not measurable. Upon inhibition of enolase by NaF, however, both 2,3-DPG and 3-PGA increased to a similar extent in fetal and adult red cells. These findings point towards differences in the pyruvate kinase (PK) reaction which is one of the rate limiting steps of glycolysis. Direct measurements revealed an over tenfold higher PK activity in fetal compared to adult red cells. This higher activity of PK will lead to a decreased concentration of 3-PGA with a consecutive fall in 2,3-DPG concentration. 3. Other factors, like a decreased glucose utilization, a decreased activity of 2,3-DPG mutase or an increased 2,3-DPG phosphatase activity could be excluded as a cause for the low 2,3-DPG concentration in fetal red blood cells. The same holds for extraerythrocytic factors like glucose concentration or pH value in fetal blood. 4. During the postnatal development of rabbits the PK activity decreased. 50 days after birth, PK activity was 20% of the fetal value but still somewhat higher than in adult erythrocytes. This change is paralleled by an increase in 2,3-DPG concentration and half saturation oxygen pressure. With respect to the synthesis of 2,3-DPG and ATP, the fetal rabbit red cell is comparable to hereditary high PK activity in human erythrocytes.

Animals↗

PubMed1993

Fractionation of erythroblasts with affinity-mediated modifications of their electrical properties using counter-current distribution.

We have previously reported the possibility of modifying the electrical properties of cells by means of their interaction with a specific ligand carrying a polyelectrolyte (Anal Biochem 200: 280-285). This selective modification of receptor-containing cells changed their partition in a charge-sensitive aqueous two-phase system. We here present the fractionation of electrically modified erythroblasts by the use of an automatic multiple-partition procedure, counter-current distribution. The cells were fractionated according to the degree of differentiation of erythroblasts as evaluated from the hemoglobin content as well as the relative activities of the two enzymes, 3-phosphoglycerate kinase and bisphospho-glycerate mutase.

Animals↗

PubMed1990

2,3-Bisphosphoglycerate, fructose, 2,6-bisphosphate and glucose 1,6-bisphosphate during maturation of reticulocytes with low 2,3-bisphosphoglycerate content.

In contrast to the species with erythrocytes of high 2,3-bisphosphoglycerate content, in the sheep the concentration of 2,3-bisphosphoglycerate decreases during maturation of reticulocytes. The decrease can be explained by the drop of the phosphofructokinase/pyruvate kinase and 2,3-bisphosphoglycerate synthase/2,3-bisphosphoglycerate phosphatase activity ratios that result from the decline of phosphofructokinase, pyruvate kinase, phosphoglycerate mutase and the bifunctional enzyme 2,3-bisphosphoglycerate synthase/phosphatase. The concentrations of fructose 2,6-bisphosphate and aldohexose 1,6-bisphosphates also decrease during sheep reticulocyte maturation in parallel to the 6-phosphofructo 2-kinase and the glucose 1,6-bisphosphate synthase activities.

2,3-Diphosphoglycerate↗