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A Peterkofsky

Publications and source records attributed to A Peterkofsky.

At least 37 records · Page 2Linked to original sources
PubMed1997

Expression, purification, and characterization of enzyme IIA(glc) of the phosphoenolpyruvate:sugar phosphotransferase system of Mycoplasma capricolum.

The gene encoding enzyme IIA(glc) (EIIA) of the phosphoenolpyruvate:sugar phosphotransferase system of Mycoplasma capricolum was cloned into a regulated expression vector. The purified protein product of the overexpressed gene was characterized as an active phosphoacceptor from HPr with a higher pI than previously described EIIAs. M. capricolum EIIA was unreactive with antibodies directed against the corresponding proteins from either Gram-positive or Gram-negative bacteria. Enzyme IIA(glc) behaved as a homogeneous, monomeric species of 16,700 Mr in analytical ultracentrifugation. The circular dichroism far-UV spectrum of EIIA reflects a low alpha-helical content and predominantly beta-sheet structural content: temperature-induced changes in ellipticity at 205 nm showed that the protein undergoes reversible, two-state thermal unfolding with Tm = 70.0 +/- 0.3 degrees C and a van't Hoff deltaH of 90 kcal/mol. Enzyme I (64,600 Mr) from M. capricolum exhibited a monomer-dimer-tetramer association at 4 and 20 degrees C with dimerization constants of log K(A) = 5.6 and 5.1 [M(-1)], respectively, in sedimentation equilibrium experiments. A new vector, capable of introducing an N-terminal His tag on a protein, was developed in order to generate highly purified heat-stable protein (HPr). No significant interaction of EIIA with HPr was detected by gel-filtration chromatography, intrinsic tryptophanyl residue fluorescence changes, titration calorimetry, biomolecular interaction, or sedimentation equilibrium studies. While Escherichia coli EIIA inhibits Gram-negative glycerol kinase activity, the M. capricolum EIIA has no effect on the homologous glycerol kinase. The probable regulator of sugar transport systems, HPr(Ser) kinase, was demonstrated in extracts of M. capricolum and Mycoplasma genitalium. Gene mapping studies demonstrated that, in contrast to the clustered arrangement of genes encoding HPr and enzyme I in E. coli, these genes are located diametrically opposite in the M. capricolum chromosome.

Chromosome Mapping↗
PubMed1997

Identification by NMR of the binding surface for the histidine-containing phosphocarrier protein HPr on the N-terminal domain of enzyme I of the Escherichia coli phosphotransferase system.

The interaction between the approximately 30 kDa N-terminal domain of enzyme I (EIN) and the approximately 9.5 kDa histidine-containing phosphocarrier protein HPr of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has been investigated by heteronuclear magnetic resonance spectroscopy. The complex is in fast exchange, permitting us to follow the chemical shift changes of the backbone NH and 15N resonances of EIN upon complex formation by recording a series of 1H-15N correlation spectra of uniformly 15N-labeled EIN in the presence of increasing amounts of HPr at natural isotopic abundance. The equilibrium association constant derived from analysis of the titration data is approximately 1.5 x 10(5) M(-1), and the lower limit for the dissociation rate constant is 1100 s(-1). By mapping the backbone chemical shift perturbations on the three-dimensional solution structure of EIN [Garrett, D. S., Seok, Y.-J., Liao, D.-I., Peterkofsky, A., Gronenborn, A. M., & Clore, G. M. (1997) Biochemistry 36, 2517-2530], we have identified the binding surface of EIN in contact with HPr. This surface is primarily located in the alpha domain and involves helices H1, H2, and H4, as well as the hinge region connecting helices H2 and H2'. The data also indicate that the active site His 15 of HPr must approach the active site His 189 of EIN along the shallow depression at the interface of the alpha and alpha/beta domains. Interestingly, both the backbone and side chain resonances (assigned from a long-range 1H-15N correlation spectrum) of His 189, which is located at the N-terminus of helix H6 in he alpha/beta domain, are only minimally perturbed upon complexation, indicating that His 189 (in the absence of phosphorylation) does not undergo any significant conformational change or change in pK(a) value upon HPr binding. On the basis of results of this study, as well as a previous study which delineated the interaction surface for EI on HPr [van Nuland, N. A. J., Boelens, R., Scheek, R. M., & Robillard, G. T. (1995) J. Mol. Biol. 246, 180-193], a model for the EIN/HPr complex is proposed in which helix 1 (residues 16-27) and the helical loop (residues 49-53) of HPr slip between the two pairs of helices constituting the alpha domain of EIN. In addition, we suggest a functional role for the kink between helices H2 and H2' of EIN, providing a flexible joint for this interaction to take place.

Bacterial Proteins↗
PubMed1997

Solution structure of the 30 kDa N-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system by multidimensional NMR.

The three-dimensional solution structure of the 259-residue 30 kDa N-terminal domain of enzyme I (EIN) of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli has been determined by multidimensional nuclear magnetic resonance spectroscopy. Enzyme I, which is autophosphorylated by phosphoenolpyruvate, reversibly phosphorylates the phosphocarrier protein HPr, which in turn phosphorylates a group of membrane-associated proteins, known as enzymes II. To facilitate and confirm NH, 15N, and 13C assignments, extensive use was made of perdeuterated 15N- and 15N/13C-labeled protein to narrow line widths. Ninety-eight percent of the 1H, 15N, and 13C assignments for the backbone and first side chain atoms of protonated EIN were obtained using a combination of double and triple resonance correlation experiments. The structure determination was based on a total of 4251 experimental NMR restraints, and the precision of the coordinates for the final 50 simulated annealing structures is 0.79 +/- 0.18 A for the backbone atoms and 1.06 +/- 0.15 A for all atoms. The structure is ellipsoidal in shape, approximately 78 A long and 32 A wide, and comprises two domains: an alpha/beta domain (residues 1-20 and 148-230) consisting of six strands and three helices and an alpha-domain (residues 33-143) consisting of four helices. The two domains are connected by two linkers (residues 21-32 and 144-147), and in addition, at the C-terminus there is another helix which serves as a linker between the N- and C-terminal domains of intact enzyme I. A comparison with the recently solved X-ray structure of EIN [Liao, D.-I., Silverton, E., Seok, Y.-J., Lee, B. R., Peterkofsky, A., & Davies, D. R. (1996) Structure 4, 861-872] indicates that there are no significant differences between the solution and crystal structures within the errors of the coordinates. The active site His189 is located in a cleft at the junction of the alpha and alpha/beta domains and has a pKa of approximately 6.3. His189 has a trans conformation about chi1, a g+ conformation about chi2, and its Nepsilon2 atom accepts a hydrogen bond from the hydroxyl proton of Thr168. Since His189 is thought to be phosphorylated at the N epsilon2 position, its side chain conformation would have to change upon phosphorylation.

Amino Acid Sequence↗
PubMed1996

The first step in sugar transport: crystal structure of the amino terminal domain of enzyme I of the E. coli PEP: sugar phosphotransferase system and a model of the phosphotransfer complex with HPr.

BACKGROUND: The bacterial phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) transports exogenous hexose sugars through the membrane and tightly couples transport with phosphoryl transfer from PEP to the sugar via several phosphoprotein intermediates. The phosphate group is first transferred to enzyme I, second to the histidine-containing phosphocarrier protein HPr, and then to one of a number of sugar-specific enzymes II. The structures of several HPrs and enzymes IIA are known. Here we report the structure of the N-terminal half of enzyme I from Escherichia coli (EIN). RESULTS: The crystal structure of EIN (MW approximately 30 kDa) has been determined and refined at 2.5 A resolution. It has two distinct structural subdomains; one contains four alpha helices arranged as two hairpins in a claw-like conformation. The other consists of a beta sandwich containing a three-stranded antiparallel beta sheet and a four-stranded parallel beta sheet, together with three short alpha helices. Plausible models of complexes between EIN and HPr can be made without assuming major structural changes in either protein. CONCLUSIONS: The alpha/beta subdomain of EIN is topologically similar to the phosphohistidine domain of the enzyme pyruvate phosphate dikinase, which is phosphorylated by PEP on a histidyl residue but does not interact with HPr. It is therefore likely that features of this subdomain are important in the autophosphorylation of enzyme I. The helical subdomain of EIN is not found in pyruvate phosphate dikinase; this subdomain is therefore more likely to be involved in phosphoryl transfer to HPr.

Amino Acid Sequence↗
PubMed1996

Importance of the region around glycine-338 for the activity of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

The gene encoding enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system from an Escherichia coli enzyme I mutant was cloned and sequenced. The mutation was shown to be a guanine to adenine transition resulting in an altered protein in which glycine-338 was replaced by aspartic acid. The enzyme I structural gene was mutated to change glycine-338 to a variety of other amino acid residues. Fermentation tests indicated that glycine-338 could be mutated to alanine with no gross loss in phosphotransferase activity, while mutation to valine, glutamic acid, aspartic acid, arginine, histidine, or asparagine led to significant loss of activity. An expression vector for enzyme I was mutated to change glycine-338 to a variety of other amino acid residues and highly purified mutant proteins were prepared. Analysis of phosphorylation of the proteins by PEP indicated that mutation of glycine-338 to alanine had little effect on phosphorylation, mutation to valine substantially decreased phosphorylation, change to histidine or arginine drastically diminished phosphorylation, and mutation to aspartic or glutamic acids abolished phosphorylation activity. Mutation at glycine-338 influences the autophosphorylation rather than the phosphoryl transfer activity of enzyme I.

Amino Acid Sequence↗
PubMed1996

Importance of the carboxyl-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system for phosphoryl donor specificity.

The first protein component of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system (PTS) is the 64-kDa protein enzyme I (EI), which can be phosphorylated by phosphoenolpyruvate (PEP) and carry out phosphotransfer to the acceptor heat-stable protein (HPr). The isolated amino-terminal domain (EIN) of E. coli EI is no longer phosphorylated by PEP but retains the ability to participate in reversible phosphotransfer to HPr. An expression vector was constructed for the production of large amounts of EIN, and conditions were developed for maximal expression of the protein. A three-column procedure is described for purification to homogeneity of EIN; a 500-ml culture yields approximately 80 mg of pure protein in about a 75% yield. Intact E. coli EI is effective in phosphotransfer from PEP to HPr from E. coli but not to the HPrs from Bacillus subtilis or Mycoplasma capricolum. Phosphotransfer from EI to enzyme IIAglc (EIIAglc) from E. coli or M. capricolum requires the intermediacy of HPr. The phosphorylated form of EIN is capable of more general phosphotransfer; it will effect phosphotransfer to HPrs from E. coli, B. subtilis, and M. capricolum as well as to EIAglc from E. coli. These studies demonstrate that the carboxyl-terminal domain of EI confers on the protein the capability to accept a phosphoryl group from PEP as well as a discriminator function that allows the intact protein to promote effective phosphoryl transfer only to E. coli HPr.

Amino Acid Sequence↗
PubMed1996

Sequence and organization of genes encoding enzymes involved in pyruvate metabolism in Mycoplasma capricolum.

The region of the genome of Mycoplasma capricolum upstream of the portion encompassing the genes for Enzymes I and IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) was cloned and sequenced. Examination of the sequence revealed open reading frames corresponding to numerous genes involved with the oxidation of pyruvate. The deduced gene organization is naox (encoding NADH oxidase)-lplA (encoding lipoate-protein ligase)-odpA (encoding pyruvate dehydrogenase EI alpha)-odpB (encoding pyruvate dehydrogenase EI beta)-odp2(encoding pyruvate dehydrogenase EII)-dldH (encoding dihydrolipoamide dehydrogenase)-pta (encoding phosphotransacetylase)-ack (encoding acetate kinase)-orfA (an unknown open reading frame)-kdtB-ptsI-crr. Analysis of the DNA sequence suggests that the naox and lplA genes are part of a single operon, odpA and odpB constitute an additional operon, odp2 and dldH a third operon, and pta and ack an additional transcription unit. Phylogenetic analyses of the protein products of the odpA and odpB genes indicate that they are most similar to the corresponding proteins from Mycoplasma genitalium, Acholeplasma laidlawii, and Gram-positive organisms. The product of the odp2 gene contains a single lipoyl domain, as is the case with the corresponding proteins from M. genitalium and numerous other organisms. An evolutionary tree places the M. capricolum odp2 gene product in close relationship to the corresponding proteins from A. laidlawii and M.genitalium. The dldH gene encodes an unusual form of dihydrolipoamide dehydrogenase that contains an aminoterminal extension corresponding to a lipoyl domain, a property shared by the corresponding proteins from Alcaligenes eutrophus and Clostridium magnum. Aside from that feature, the protein is related phylogenetically to the corresponding proteins from A. laidlawii and M. genitalium. The phosphotransacetylase from M. capricolum is related most closely to the corresponding protein from M. genitalium and is distinguished easily from the enzymes from Escherichia coli and Haemophilus influenzae by the absence of the characteristic amino-terminal extension. The acetate kinase from M. capricolum is related evolutionarily to the homologous enzyme from M. genitalium. Map position comparisons of genes encoding proteins involved with pyruvate metabolism show that, whereas all the genes are clustered in M. capricolum, they are scattered in M. genitalium.

Acetate Kinase↗
PubMed1995

Structural evidence for the evolutionary divergence of mycoplasma from gram-positive bacteria: the histidine-containing phosphocarrier protein.

BACKGROUND: The three-dimensional structures of histidine-containing phosphocarrier protein (HPr), a member of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), have been determined from Gram-negative and Gram-positive bacteria. The structure of HPr reported here for Mycoplasma capricolum is the first protein structure to be determined for this class of organism. Comparative structural studies with the bacterial proteins highlight sequence-structure correlations relevant to proposals about the evolutionary origin of mycoplasmas. RESULTS: The crystal structure of HPr from M. capricolum has been determined and refined at 1.8 A resolution, revealing the same overall fold as that of other HPrs of known structure. However, mycoplasma HPr resembles HPrs from Gram-positive bacteria more closely than those from Gram-negative bacteria. As in HPrs from Bacillus subtilis and Escherichia coli, the phosphoryl group carrier (His15) forms the N-terminal cap of a helix, but in contrast to the other crystal structures, the side chain of the adjacent Arg17 is conformationally disordered. A sulfate ion interacts with Ser46, a residue known to be phosphorylated in a regulatory manner. CONCLUSIONS: The greater degree of structural similarity of the M. capricolum HPr to HPrs from Gram-positive rather than Gram-negative bacteria is consistent with the proposal that mycoplasma evolved from Gram-positive bacteria. The proposal that no major conformational transition is required for phosphorylation of the active-site histidine is reinforced by comparing the crystal structures with and without an anion in the active site. The conformational disorder of the Arg17 side chain suggests that its guanidinium group does not have to form specific interactions with other protein groups before phosphorylation at His15. The association of a sulfate ion with Ser46 serves as a model for HPr(Ser46-P). As there is no evidence of a conformational change accompanying Ser46 phosphorylation, the inhibitory effect of this event may be attributable to altered surface electrostatics.

Amino Acid Sequence↗
PubMed1995

The Escherichia coli adenylyl cyclase complex: requirement of PTS proteins for stimulation by nucleotides.

GTP, as well as other nucleoside triphosphates, stimulates the activity of Escherichia coli adenylyl cyclase in permeable cells; the stimulatory effect is lost when the cells are disrupted by passage through a French pressure cell. These data suggested that the allosteric regulation by GTP of adenylyl cyclase activity requires an interaction of the enzyme with other protein factors. Strains deleted for genes encoding proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) failed to show an activity stimulation by GTP. With a view to localizing the site of interaction of GTP with the adenylyl cyclase complex, a variety of studies using purified PTS proteins were performed using the photoaffinity labeling reagent, 8-azidoGTP. These studies showed that 8-azidoGTP bound specifically to HPr. A species specificity study showed that the photoaffinity reagent labeled E. coli HPr but not HPr proteins from Mycoplasma capricolum or Bacillus subtilis. A variety of site-directed mutations of E. coli HPr were evaluated for interaction with GTP by photoaffinity labeling as well as by nuclear magnetic resonance; the results of these studies indicate that the lysine residues at positions 24 and 27, serine-46, the threonine at position 36, and the aspartate at position 69 are important for the binding of GTP to HPr. Molecular modeling has been used to formulate a model for the binding of GTP to HPr involving electrostatic interaction of the phosphate groups of the nucleotide with the side chains of lysine residues 27 and 45 and serine-43, interaction of the sugar with serine-46, and interaction of the base with lysine-24. From these data, it is hypothesized that the binding of GTP to HPr is required for the GTP-dependent stimulation of the activity of the adenylyl cyclase complex.

Adenylyl Cyclases↗
PubMed1995

A dual mechanism for regulating cAMP levels in Escherichia coli.

In Escherichia coli, inorganic orthophosphate regulates cAMP levels by acting at two separate loci. First, adenylyl cyclase activity measured in permeabilized cells of E. coli is substantially stimulated by physiological concentrations of inorganic phosphate. This stimulation does not require the presence of cAMP phosphodiesterase activity. Second, measurements of cAMP phosphodiesterase activity in permeabilized cells show a dose-dependent inhibition of that activity by inorganic orthophosphate. A model is proposed in which inorganic orthophosphate serves as a multifaceted regulator of cAMP levels by both stimulating synthesis and inhibiting degradation of the nucleotide.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Organization, sequence and regulation of expression of the murine Hoxa-7 gene.

The genomic sequence of Hoxa-7 (encoding the HOXa-7 homeobox protein), including the coding region (0.7 kb), flanked by a 5'-upstream region (2.8 kb), a 3'-downstream region (1 kb) and interrupted by an intron (995 bp), was determined. Northern blot analysis indicated the transcript size of Hoxa-7 to be 2.1-2.4 kb. Reverse transcription-PCR and primer extension analysis established the 5'-boundary of the mRNA to be in the region 1166 nt upstream from the start codon. Transient transfection of various Hoxa-7::cat constructs in NIH 3T3 cells was used to characterize the transcriptional activity of the 5'-flanking region of the gene. Constructs containing 544, 274 and 71 bp of the region upstream from the transcription start point (tsp) exhibited 78, 203 and 407%, respectively, of the activity shown by a control construct containing 739 bp of the upstream region. These data suggested the presence of negative regulatory elements in the region from 544 to 71 bp upstream from the tsp.

3T3 Cells↗
PubMed1995

Overproduction and purification of the Mycoplasma capricolum phosphocarrier protein, HPr, of the phosphoenolpyruvate: sugar phosphotransferase system.

The gene (ptsH) for the phosphocarrier protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system from Mycoplasma capricolum was previously cloned and sequenced. We present here the results of experiments in which the ptsH gene was cloned into a vector for high level expression in Escherichia coli of the phosphocarrier protein. Conditions were developed for overproduction and purification of HPr by a two-column procedure. The purified protein, analyzed by Edman degradation and mass spectrometry, was found to have been processed by removal of the N-terminal methionine residue. Examination of the purified protein by gel electrophoresis under isoelectric focusing conditions revealed that it has an unusually high isoelectric point.

Amino Acid Sequence↗
PubMed1994

Importance of the region around lysine 196 for catalytic activity of adenylyl cyclase from Escherichia coli.

Escherichia coli adenylyl cyclase contains no sequence that corresponds to the previously defined ATP/GTP binding consensus (A,G)XXXXGK(S,T). Using a search for lysine residues located adjacent to glycine residues, three regions that were possible candidates for part of the ATP binding site were identified. These were the residues located at positions 59, 90, and 196. A plasmid vector capable of overexpressing the cya gene under the control of the lambda PL promoter was mutated at these three loci to convert those lysine residues to methionine. Assays for catalytic activity of the mutated hyperexpressed proteins revealed that only the mutation at position 196 led to loss of activity. Photoaffinity labeling experiments using 8-azido-ATP provided evidence that the loss of activity was associated with a loss of the capability of the enzyme to bind ATP. A further series of replacement mutations in the hyperexpression vector was created at position 196. Assays of the adenylyl cyclase activity of the mutated proteins showed that replacement of lysine 196 by arginine led to minimal change in the activity. Replacements by histidine, glutamine, or glutamic acid resulted in approximately 10-20-fold reductions in the activity; replacements by methionine, isoleucine, or aspartic acid resulted in total loss of activity. When the mutated forms of the cya gene were expressed under the control of the cya promoter, the activity of the wild-type protein was higher than that of all the mutants, including the arginine replacement mutant. All of the mutants that retained activity also retained the capability of adenylyl cyclase to be stimulated by either inorganic orthophosphate or GTP. A helical wheel analysis of the region of adenylyl cyclase around lysine 196 revealed a structure compatible with an amphipathic helix with one face enriched with basic amino acid residues. Assays for adenylyl cyclase activity of a series of replacement mutations of residues on the hydrophilic face of the helix (R188I, R192I, G195I) as well as on the hydrophobic face (R197I) indicated that the R188I, G195I, and K196I replacement mutants were inactive, and R192I was approximately 30% as active as the wild-type, while the R197I mutant was equivalent to the wild-type control. A model is suggested for a unique binding motif in E. coli adenylyl cyclase in which there is a repetition of 3 basic residues on one face of a helix where there is an interaction with the three phosphate groups of ATP.

Adenosine Triphosphate↗
PubMed1994

Unique dicistronic operon (ptsI-crr) in Mycoplasma capricolum encoding enzyme I and the glucose-specific enzyme IIA of the phosphoenolpyruvate:sugar phosphotransferase system: cloning, sequencing, promoter analysis, and protein characterization.

The region of the genome of Mycoplasma capricolum encompassing the genes for Enzymes I and IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) was cloned and sequenced. Examination of the sequence revealed a unique arrangement of the pts operon. In all other bacterial species characterized thus far, the gene encoding Enzyme I (ptsI) in the pts operon is located immediately downstream of the gene (ptsH) encoding HPr, a general energy coupling protein of the PTS. In M. capricolum, ptsH and ptsI reside on 2 distinct operons at separate loci on the chromosome (Zhu PP, Reizer J, Reizer A, Peterkofsky A, 1993, J Biol Chem 268:26531-26540). In the present work, it is shown that the Mycoplasma Enzyme I gene is preceded by an open reading frame homologous to the product of the Escherichia coli kdtB gene and is followed by the gene (crr) encoding Enzyme IIAglc. Northern blot analysis indicated that ptsI and crr constitute a dicistronic operon that includes an independent promoter for the crr gene. Primer extension studies established the transcription start sites for the ptsI and crr genes. The products of the ptsI and crr genes are homologous to previously sequenced Enzymes I and IIAglc proteins but are more similar to the counterpart proteins from gram-positive than to those from gram-negative organisms. The deduced amino acid sequence of the Mycoplasma Enzyme I shows that it differs from other Enzymes I by having fewer acidic amino acids and more basic, amidated, and aromatic amino acids. The deduced amino acid sequence of the Mycoplasma Enzyme IIAglc indicates that it is the shortest (154 residues) of the proteins in this class and it is the only Enzyme IIAglc with a tryptophan and a cysteine residue. In vitro sugar phosphorylation studies with extracts from E. coli and Bacillus subtilis and purified proteins indicated that the Mycoplasma HPr is not a phosphoacceptor from the E. coli Enzyme I, whereas the Mycoplasma Enzyme IIAglc accepts and transfers phosphate from both E. coli and B. subtilis PTS components.

Amino Acid Sequence↗
PubMed1994

Identification of the N-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system produced by proteolytic digestion.

The phosphoenolpyruvate:sugar phosphotransferase system of bacteria plays an important role in the concomitant uptake and phosphorylation of numerous sugars. The first protein in the pathway of phosphotransfer of the phosphoenolpyruvate:sugar phosphotransferase system is Enzyme I. It has been shown that a stable N-terminal domain can be produced by treatment of the purified protein with various proteolytic enzymes. We show here that the region from glutamate-252 to leucine-264 is accessible to proteolysis resulting in N-terminal cores ranging from M(r) 27521 to 28799.

Amino Acid Sequence↗
PubMed1993

Unique monocistronic operon (ptsH) in Mycoplasma capricolum encoding the phosphocarrier protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system. Cloning, sequencing, and characterization of ptsH.

The region of the genome of Mycoplasma capricolum encompassing the gene (ptsH) encoding HPr, a general energy-coupling protein of the phosphoenolpyruvate:sugar phosphotransferase system, was cloned and sequenced. Examination of the sequence revealed a unique arrangement of the ptsH gene. In all other bacterial species characterized thus far, the ptsH gene is part of a polycistronic operon that includes the gene (ptsI) encoding Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system; the M. capricolum ptsH gene is part of a monocistronic operon that is situated between two open reading frames unrelated to phosphoenolpyruvate:sugar phosphotransferase system function. The gene immediately upstream of ptsH codes for a helicase, and the open reading frame immediately downstream of ptsH, although not homologous to any previously identified protein, contains a signature sequence characteristic of [C-5] cytosine-specific DNA methylases. The product of the ptsH gene has characteristics similar to the HPr protein produced by Gram-positive organisms: it has a greater sequence similarity to HPrs of Gram-positive bacteria than to those of Gram-negative organisms, it is phosphorylated by a protein kinase derived from Gram-positive organisms, and it complements sugar phosphorylation activity in Gram-positive extracts. The high calculated isoelectric point (pI = 9.18) and the absence of glutamate residues in the C-terminal region distinguish the M. capricolum HPr from all previously described HPrs.

Amino Acid Sequence↗
PubMed1993

The Escherichia coli adenylyl cyclase complex: stimulation by GTP and other nucleotides.

Escherichia coli cells permeabilized by treatment with low concentrations of toluene contain an adenylyl cyclase activity that can be stimulated 3.6-7.6-fold by GTP. The stimulatory effect of GTP is maximal at concentrations of the nucleotide in the physiological range (above 0.7 mM). Studies of the dependence of velocity on substrate (ATP) concentration indicate that the velocity vs. substrate plots are sigmoid in the absence of GTP but hyperbolic in the presence of GTP, suggesting an allosteric regulatory site that can be occupied by either ATP or GTP. Replacement of ATP by AMPPNP as substrate results in velocity vs. substrate plots that are hyperbolic in the absence or presence of GTP, although GTP increases the Vmax by a factor of 2.2; these findings indicate that AMPPNP specifically occupies the substrate site and GTP exclusively occupies the regulatory site. A test of the capacity of other guanine nucleotides to stimulate adenylyl cyclase activity showed that 2'-deoxy-GTP was almost as effective as GTP, but that GDP, GMP, ppGpp, and 3',5'-cGMP were not stimulatory effectors; GTP-gamma-S and GMPPNP stimulated adenylyl cyclase activity but to a lesser degree than did GTP. In addition to the previous indication that ATP can occupy the regulatory site on adenylyl cyclase, it was found that CTP and UTP were potent stimulators. Thus, all the naturally occurring RNA precursor nucleoside triphosphates are capable of stimulating adenylyl cyclase activity. In contrast, PPPi inhibits adenylyl cyclase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗