Cloning and expression of the gene for the active PPi-dependent phosphofructokinase of Entamoeba histolytica.

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) from Entamoeba histolytica (HM-1) was purified from trophozoites. Oligonucleotide probes based on partial amino acid sequence were used to clone and sequence the gene and the cDNA of the enzyme. The molecular mass of the subunit was greater than, and the derived sequence significantly different from, that of the product of the PPi-PFK gene previously cloned from E. histolytica [Huang, Albach, Chang, Tripathi and Kemp (1995) Biochim. Biophys. Acta 1260, 215-217; Bruchhaus, Jacobs, Denart and Tannich (1996) Biochem. J. 316, 57-63]. The sequence identity between the two proteins was 17%. The sequence bore greater identity with the more phylogenetically advanced plant PPi-PFKs than with bacterial PPi-PFKs. The cloned cDNA was expressed and the protein purified. The kinetic properties were identical with those of the enzyme isolated from the organism. Furthermore, the specific activity was more than three orders of magnitude higher than that described for the product of the previously cloned E. histolytica PFK gene [Bruchhaus et al. (1996)]. The pH-dependence and apparent substrate affinities of the cloned enzyme were identical with those of the PPi-PFK in trophozoite extracts, indicating that the product of the cloned gene accounts for most if not all of the PFK activity in E. histolytica trophozoites.

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: revised amino acid sequence, site-directed mutagenesis, and microenvironment characteristics of cysteines 365 and 458.

Two cysteine residues in phosphoenolpyruvate (PEP) carboxykinase from Saccharomyces cerevisiae [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] the modification of which leads to enzyme inactivation have been subjected to site-directed mutagenesis. PEP carboxykinase is inactivated by alkylation of Cys365 or Cys458; however, mutation of either or both of these residues to serine has little effect on the enzymatic activity. These results eliminate any possible catalytic function for these cysteinyl residues. In the course of this work, discrepancies in the published nucleotide sequence of the S. cerevisiae PEP carboxykinase gene were detected that alter the deduced amino acid sequence. Several of these discrepancies were verified through the sequencing of proteolytic peptides. Our results indicate that the protein corresponds to a 549 amino acid polypeptide and that the positions previously assigned to Cys364 and Cys457 correspond to Cys365 and Cys458. The individual reactivities and the microenvironment characteristics around these sulfhydryl groups were investigated by their selective modification with the fluorescent reagent N-(1-pyrenyl)maleimide (PyM). Our findings indicate that Cys458 is 7-fold more reactive toward the sulfhydryl-directed probe than Cys365, while quenching experiments of PyM-labeled mutant enzymes suggest that the former residue is located in a region more accessible to water than the latter.

Structure and expression of the cDNA for the C isozyme of phosphofructo-1-kinase from rabbit brain.

Rabbit brain contains three phosphofructo-1-kinase (PFK) isozymic subunits designated A, B, and C. The primary structures of the first of these two isozyme types have been determined previously. The isozyme C of rabbit brain was isolated by immunoaffinity chromatography and subjected to proteolytic and chemical digestion. A large number of peptides were sequenced, the total number of amino acids identified being equal to about 80% of the total structure. The sequence of the cDNA derived from brain mRNA for C isozyme was determined from polymerase chain reaction fragments synthesized using oligonucleotides designed on the basis of the peptide sequences. The deduced size of the C isozyme was 86,371 Da, slightly larger than PFKs described previously. The amino acid sequence identity with the rabbit A isozyme was 68.9% and a range of identity to other sequenced mammalian PFKs was 67-69%. Using these data plus previously published data on chemical modification, assignments of the 6 organic ligand binding sites of PFK were inferred. The full-length cDNA was cloned into and expressed in Escherichia coli. Phosphofructokinase C was purified to homogeneity from the bacterial extracts.

Identification of reactive vicinal cysteines in Saccharomyces cerevisiae (ATP) and cytosolic rat liver (GTP) phospho enol pyruvate carboxykinases.

Saccharomyces cerevisiae (ATP) and cytosolic rat liver (GTP) phospho enol pyruvate carboxykinases (EC 4.1.1.49/32) have been labeled with N-(1-pyrenyl)-iodoacetamide. Reagent incorporation was completely prevented by the presence of the respective nucleoside diphosphate plus MnCl2. Under appropriate conditions, 2 mol of reagent per mol of enzyme subunit were incorporated. The fluorescence spectra of the labeled proteins showed the pyrene excimer emission band. The pyrenyl-derivatized enzymes were digested with trypsin after carboxymethylation, and two labeled peptides were isolated for each carboxykinase upon reverse-phase high-performance liquid chromatography. Automated Edman degradation of the labeled peptides indicated that cysteines 364 and 457 (yeast enzyme), and cysteines 288 and 413 (rat enzyme) were labeled with the fluorescence SH-specific reagent. The relative reactivity of these residues was characterized. Labeling experiments utilizing the 5,5'-dithiobis(2-nitrobenzoate)-oxidized enzymes suggested that the reactive SH-groups occupy a vicinal position in the tertiary structure of the proteins, probably in the nucleotide-binding region.

Identification of critical lysyl residues in the pyrophosphate-dependent phosphofructo-1-kinase of Propionibacterium freudenreichii.

Pyrophosphate-dependent 6-phosphofructo-1-kinase (PPi-PFK) from Propionibacterium freudenreichii was inactivated by low concentrations of the lysine-specific reagent pyridoxal phosphate (PLP) after sodium borohydride reduction. The substrates fructose 6-phosphate and fructose 1,6-bisphosphate protected against inactivation whereas inorganic pyrophosphate had little effect. An HPLC profile of a tryptic digest of PPi-PFK modified at low concentrations of PLP showed a single major peak with only a small number of minor peaks. The major peak peptide was isolated and sequenced to obtain IGAGXTMVQK, where X represents a modified lysine residue, corresponding to Lys-315. Lys-315 was protected from reaction with PLP by fructose 1,6-bisphosphate. As indicated by HPLC maps of PPi-PFK modified with varying concentrations of PLP, a direct correlation was observed between activity loss and the modification of Lys-315. Two of the minor peptide peaks were shown to contain Lys-80 and Lys-85, which were modified in a mutually exclusive manner. Partial protection against modification of these two residues was provided by MgPPi. The data were used to adjust the sequence alignment of the Propionibacterium enzyme with that of ATP-dependent PFK of Escherichia coli to identify homologous residues in the substrate binding site. It is suggested that Lys-315 interacts with the 6-phosphate of fructose 6-phosphate and that Lys-80 and -85 may be located near the pyrophosphate binding site.

ATP-dependent Saccharomyces cerevisiae phospho enol pyruvate carboxykinase: isolation and sequence of a peptide containing a highly reactive cysteine.

Saccharomyces cerevisiae phospho enol pyruvate carboxykinase (EC 4.1.1.49), inactivated by N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine, incorporated 0.95 mol of the fluorescent moiety per mol of enzyme subunit. Reagent incorporation was completely protected by the presence of ADP plus MnCl2. The labeled protein was digested with trypsin after carboxymethylation. Two labeled peptides were isolated by reverse-phase high-performance liquid chromatography and were sequenced by gas-phase automatic Edman degradation. Both peptides contained overlapping amino acid sequences from Asn-358 to Lys-375, thus identifying Cys-364 as the reactive amino acid residue. The position of the target amino acid residue is immediately preceding a putative phosphoryl-binding sequence proposed for some nucleotide-binding proteins.

Cloning, sequencing, and expression of pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii.

Pyrophosphate-dependent 6-phosphofructo-1-kinase (PPi-PFK) from Propionibacterium freudenreichii is a non-allosteric enzyme with properties dissimilar to those of other described phosphofructokinases. The enzyme was cloned into pBluescript, sequenced, and expressed in Escherichia coli at levels 15 times higher than those observed in Propionibacterium. The gene consists of 1215 bases which code for a protein of 404 amino acids and a mass of 43,243 daltons. High G + C in the codon usage (66%) of the gene is consistent with the classification of Propionibacterium in the High-G + C subdivision of the Gram-positive bacteria. While showing no sequence identity to the non-allosteric ATP-dependent phosphofructokinase of E. coli, alignments of the amino acid sequence with other PFKs reveal degrees of identities among the amino halves of the proteins, from 26% between the Propionibacterium and potato PPi-PFKs, and 29% between Propionibacterium and E. coli ATP-PFKs. These levels of identities indicate that the amino halves of these proteins are homologous. Identities between the carboxyl half of Propionibacterium PFK and carboxyl halves of other sequences are below 20%, suggesting that the carboxyl half is not homologous. Despite the poor conservation, most of the residues that take part in the binding of fructose-6-P or Mg-PPi could be readily identified by analogy to the structure of the E. coli PFK. Both the fructose-6-P and ATP-binding sites are conserved, indicating that PPi binds to the homologous site of the E. coli ATP-binding site.

Spinach cytosolic fructose-1,6-bisphosphatase. Purification, enzyme properties and structural comparisons.

Cytosolic fructose-1,6-bisphosphatase from spinach leaves was purified to homogeneity and characterized. The pure enzyme has a subunit mass of 38 kDa, its Km values for fructose 1,6-bisphosphate and Mg2+ are 1.5 microM and 260 mM, respectively, and its Vmax is 110-120 units/mg. It is inhibited by fructose 2,6-bisphosphate and AMP with Ki values of 0.07 microM and 120 microM, respectively. About 90% of the primary structure of the spinach cytosolic fructose-1,6-bisphosphatase has been determined by amino-acid sequencing. The sequence data demonstrate that the cytosolic enzyme lacks the sequence insert characteristic of chloroplast fructose-1,6-bisphosphatase. The data include also the sequences of peptides containing all seven cysteine residues. Only two of the seven cysteines are conserved between the two isozymes, none of which is believed to be involved with the light regulation of the chloroplast enzyme. Sequence comparisons between the spinach cytosolic enzyme and gluconeogenic fructose-1,6-bisphosphatases from other species reveal similarity ranging over 47-54%, which is higher than the 40-45% similarity between the chloroplast enzyme and gluconeogenic fructose-1,6-bisphosphatases. However, similarity between these isozymes and Escherichia coli fructose-1,6-bisphosphatase are 44% and 47% for the cytosolic and chloroplast enzymes, respectively. Similarity between the cytosolic and chloroplast counterparts is 52%, indicating wide divergence between these two fructose-1,6-bisphosphatases.

Partial amino acid sequence of fructose-1,6-bisphosphatase from the blue-green algae Synechococcus leopoliensis.

Purified fructose-1,6-bisphosphatase from the cyanobacterium Synechococcus leopoliensis was S-carboxymethylated and cleaved with trypsin. The resulting peptides were purified by reversed-phase high performance liquid chromatography and the amino acid sequence of six of the purified peptides was determined by gas-phase microsequencing. The results revealed sequence homology with other fructose-1,6-bisphosphatases. The obtained sequence data provides information required for the design of oligonucleotide hybridization probes to screen existing libraries of cyanobacterial DNA. The determination of the amino acid sequence of cyanobacterial proteins may yield important information with respect to the endosymbiotic theory of evolution.

The sites of phosphorylation of rabbit brain phosphofructo-1-kinase by cyclic AMP-dependent protein kinase.

The three isozymic subunits of phosphofructo-1-kinase present in rabbit brain and designated A, B and C were phosphorylated in vitro by cyclic AMP-dependent protein kinase with 32P-labeled ATP. Limited digestion of the labeled enzymes with trypsin or with Staphylococcus aureus V8 proteinase led to the solubilization of radiolabeled peptides derived from the three isozymic subunits. Limited digestion by V8 proteinase was accompanied by a slight reduction in the apparent sizes of the subunits, indicating that the phosphorylated sites are located near either the amino or carboxyl termini of the protein. V8 proteinase digestion led to no change in the maximal activity of the enzyme but did abolish sensitivity to ATP inhibition. The phosphopeptides of the tryptic and the V8 digests were purified by chromatography and their amino acid sequences were determined and compared to the previously established sequence from rabbit muscle isozyme A. PFK-A E H I S R K R S G E A T V PFK-B H V T R R S L S M A K G F PFK-C V S A S P R G S Y R K F L In each instance, the phosphorylated serine, underlined in the above sequences, was found to be one or two residues toward the C-terminus of one or more basic residues. No other similarities in structure were noted.

Comparative amino acid sequence of fructose-1,6-bisphosphatases: identification of a region unique to the light-regulated chloroplast enzyme.

Chloroplast fructose-1,6-bisphosphatase (Fru-P2-ase) is an essential enzyme in the photosynthetic pathway of carbon dioxide fixation into sugars. The properties of the chloroplast enzyme are clearly distinct from cytosolic gluconeogenic Fru-P2-ases. Light-dependent activation by way of a ferredoxin/thioredoxin system and insensitivity to AMP inhibition are distinctive characteristics of the chloroplast enzyme. However, the chloroplast enzyme shows a high degree of amino acid sequence similarity to gluconeogenic Fru-P2-ases. Sequence data reported for a total of 285 residues (approximately 75% of the structure) of the spinach chloroplast enzyme reveals a 46% amino acid sequence identity with pig kidney Fru-P2-ase. We now report the amino acid sequence of a region consisting of 46 additional residues. This region is located near the middle of the primary structure of the enzyme and it includes a 16-residue insert not present in other Fru-P2-ases. This sequence insert has two cysteines separated by only 4 amino acid residues (Cys-Val-Val-Asn-Val-Cys), a characteristic feature of at least three other enzymes containing redox-active cysteines. It appears likely that this region of chloroplast Fru-P2-ase is involved in light-dependent activation.

Spinach chloroplast fructose-1,6-bisphosphatase: identification of the subtilisin-sensitive region and of conserved histidines.

Chloroplast fructose-1,6-bisphosphatase (FbPase) is an essential enzyme in the photosynthetic pathway of carbon dioxide fixation into sugars. The properties of the chloroplast enzyme are clearly distinct from those of cytosolic gluconeogenic FbPases. Light-dependent activation via a ferredoxin/thioredoxin system and insensitivity to inhibition by AMP are unique characteristics of the chloroplast enzyme. However, preliminary amino acid sequence data (78 residues) have demonstrated that a significant degree of amino acid sequence similarity exists between spinach chloroplast and mammalian gluconeogenic fructose-1,6-bisphosphatase [Harrsch, P.B., Kim, Y., Fox, J.L., & Marcus, F. (1985) Biochem. Biophys. Res. Commun. 133, 520-526]. In the present study, we have identified two structural features of spinach chloroplast FbPase that appear to be common to all FbPases. These include (a) the presence of a protease-sensitive area located in a region equivalent to residues 51-71 of mammalian FbPases and (b) the recognition of two conserved histidine residues, equivalent to histidines-253 and -311 of the mammalian enzymes. In addition, we have obtained sequence information accounting for more than three-fourths of the primary structure of spinach chloroplast FbPase. The high degree of homology observed between the chloroplast enzyme and gluconeogenic FbPases suggests a common evolutionary origin for all fructose-1,6-bisphosphatases in spite of their different functions and modes of regulation.

Identification of highly reactive cysteinyl and methionyl residues of rabbit muscle phosphofructokinase.

The reactivity of the 16 thiol groups of rabbit skeletal muscle phosphofructokinase has been studied extensively over the past 20 years. Several of these thiols show high reactivity with a variety of reagents, display differential reactivity in the presence of allosteric ligands and substrates, and appear to be important to function because their modification changes activity and regulatory properties. In the present study, the location in the primary structure of several highly reactive thiol groups has been established by reaction with [14C]iodoacetate. In the course of these studies, 2 methionyl residues that are located at or near proposed ligand-binding sites are readily carboxymethylated by iodoacetate. In addition to confirming the presence of the most reactive thiol group at sequence position 88, a thiol protected from reaction by the presence of fructose-6-P and cyclic AMP has been found at position 169. Cysteine 169 is close to a residue important to the binding of fructose-6-P in the homologous structure from Bacillus stearothermophilis phosphofructokinase. The modification of Cys-169 brings about extensive, but not total, loss of activity. Another cysteine, at position 232, was found to be highly reactive also. Substrate provided partial protection against carboxymethylation at this position. Carboxymethylation of enzyme restricted to methionines 74 and 173 brought about no changes in the total activity or in the ATP inhibition profile of the enzyme. This is significant since position 74 was projected on the basis of the homologous procaryotic structure to be important in the binding of nucleotide to the allosteric site.

Amino acid sequence at the citrate allosteric site of rabbit muscle phosphofructokinase.

Previously, this laboratory has demonstrated [Colombo, G., & Kemp, R. G. (1976) Biochemistry 15, 1774-1780] that under appropriate conditions the citrate inhibitory binding site of rabbit skeletal muscle phosphofructokinase can be covalently modified by using pyridoxal phosphate and sodium borohydride. In the current study, phosphofructokinase was modified by [3H]pyridoxal phosphate and sodium borohydride with or without the addition of citrate to protect the ligand binding site. The modified proteins were digested with trypsin, and the peptides were separated by high-pressure liquid chromatography. A comparison of the tryptic chromatographic profiles showed that while the label was broadly distributed among nine peaks in the elution profile of the enzyme modified in the presence of the protective ligand, a single peptide contained 70% of the total radioactivity of the enzyme modified in the absence of citrate. This peptide was presumed to contain at least part of the citrate inhibitory site of the enzyme. The sequence of the peptide was determined and shown to match with positions 528-536 of phosphofructokinase with the modified residue being Lys-529. A comparison of the sequence with that of procaryotic phosphofructokinase indicated that a homologous residue in the enzyme from Bacillus stearothermophilis is critical to an allosteric site. A second peptide that was the most abundant labeled peptide in the digest of the enzyme modified in the presence of citrate was found to be identical with the second most abundant peptide of the digest from the unprotected enzyme. This peptide corresponded to residues 681-692 with the lysine at position 684 being the site of phosphopyridoxylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and sequence determination of an active site peptide of rabbit muscle pyruvate kinase.

Rabbit muscle pyruvate kinase was inactivated by 2', 3'-dialdehyde ADP with the incorporation of one molecule of reagent per enzyme subunit. The inactivated protein was digested with trypsin after reduction and carboxymethylation. The labeled peptide was isolated by gel filtration and further purified by HPLC. The peptide was sequenced both by liquid-phase and gas-phase automatic Edman degradation. A 34-residue peptide was obtained. This peptide is identical to a tryptic peptide labeled with trinitrobenzenesulfonate, isolated and sequenced by Johnson et al. (Biochem. Biophys. Res. Commun. (1979) 90, 525-530) from bovine muscle pyruvate kinase. Available evidence suggests that dialdehyde ADP labels the enzyme at the same lysine in position 25 of the peptide, as found by Johnson et al. The high homology between the isolated peptide and regions of other pyruvate kinases from low to high eukaryotes supports the idea that this peptide is related to the enzyme active site.

A subpopulation of spinach chloroplast tRNA genes does not require upstream promoter elements for transcription.

We have identified a class of spinach plastid tRNA genes which do not require 5' upstream promoter elements for their expression in a chloroplast transcription system. The 5' DNA sequences flanking the trnR1 and trnS1 coding regions have little or no homology to previously characterized chloroplast promoter sequences. The deletion of the 5' DNA sequences from these genes to positions close to the start of the coding regions has little effect on their transcription in vitro. In addition, a synthetic DNA fragment homologous to the 5' region of trnS1 does not support the transcription of the promoter (-) trnM2 mutant 51 in a promoter/trnM2-51 fusion assay. In a dicistronic construct the wild type trnS1 gene does not support transcription of trnS1 transcription occurs immediately following the 3' end of the coding region. Both trnS1 and trnR1 compete with trnM2 for the same chloroplast RNA polymerase and/or common transcription factors.

Binding of hexose bisphosphates to muscle phosphofructokinase.

On the basis of kinetic activation assays, the apparent affinity of muscle phosphofructokinase for fructose 2,6-bisphosphate was about 9-fold greater than that for fructose 1,6-bisphosphate, which in turn was about 10 times higher than that for glucose 1,6-bisphosphate. Equilibrium binding experiments showed that both fructose bisphosphates bind to phosphofructokinase with negative cooperativity; the affinity for fructose 2,6-bisphosphate was about 1 order of magnitude greater than the affinity for fructose 1,6-bisphosphate. Binding of fructose 2,6-bisphosphate to phosphofructokinase was antagonized by fructose 1,6-bisphosphate and glucose 1,6-bisphosphate and vice versa. Both fructose bisphosphates promoted aggregation of the enzyme to higher polymers as indicated by sucrose density gradient centrifugation. Other indicators of phosphofructokinase conformation such as thiol reactivity and maximum activation of in vitro phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase gave identical results in the presence of fructose 2,6-bisphosphate, fructose 1,6-bisphosphate, or glucose 1,6-bisphosphate, indicating a common conformation is produced by all three ligands. It is concluded that the sugar bisphosphates bind to a single site on the enzyme.

Studies on the phosphorylation of muscle phosphofructokinase.

Phosphorylation of rabbit skeletal muscle phosphofructokinase by the catalytic subunit of cyclic AMP-dependent protein kinase occurs with a Km of about 230 microM and Vmax approaching that seen with histone as a substrate. The rate of phosphorylation of phosphofructokinase by protein kinase is increased by allosteric activators of phosphofructokinase, whereas inhibitors of phosphofructokinase inhibit the phosphorylation. Inhibitors and activators change Vmax but not Km. The site of phosphorylation is a serine residue that is the sixth amino acid from the carboxyl terminus. Limited proteolysis by trypsin releases an octapeptide from the carboxyl terminus and a brief exposure to subtilisin releases a dodecapeptide from the carboxyl end. The sequence of the dodecapeptide is His-Ile-Ser-Arg-Lys-Arg-Ser(P)-Gly-Glu-Ala-Thr-Val. Phosphofructokinase isolated from a rabbit injected 18 h prior to killing with [32P]PO4 contained covalently bound radioactive phosphate. Approximately 80% of the phosphate was released in a trichloroacetic acid-soluble form following limited proteolysis by trypsin, under which conditions the enzyme remained with a monomer size of about 80,000 daltons. The position of elution from Sephadex G-25 of the phosphopeptide was identical with that found following limited trypsin proteolysis of in vitro labeled enzyme. Migration of the phosphopeptides on thin layer cellulose chromatography was also identical. We conclude that at least 80% of the radioactive phosphate introduced within 18 h of an intravenous injection of [32P]PO4 is found at the same site as that introduced by phosphorylation with the catalytic subunit of cyclic AMP-dependent protein kinase.