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W Hengstenberg

Publications and source records attributed to W Hengstenberg.

At least 73 records · Page 4Linked to original sources
PubMed1982

Phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus: factor IIIlac, a trimeric phospho-carrier protein that also acts as a phase transfer catalyst.

Factor IIIlac (FIII) consists of three identical subunits. It could be shown that each of the subunits carries a phosphoryl group upon phosphorylation (P-FIII) with phosphoenolpyruvate (PEP), enzyme I, and histidine-containing phospho-carrier protein (HPr). The phosphoryl group is bound to a histidyl residue in P-FIII. Each subunit of FIII contains four histidyl residues. After tryptic cleavage a peptide was isolated that contained one other histidyl residue besides the active center histidine. By further cleavage of the peptide T-2 with V-8 Staphylococcus aureus protease it could be shown that His-19 in the sequence of the peptide T-2 is the active center histidine. Another peptide (1-38), caused by incomplete tryptic cleavage, could be isolated. It inhibited the phospho-transfer reaction from PEP to the sugar molecule at the step of factor III-enzyme II recognition. It competes with factor III for the binding site of enzyme II, the membrane component. It is a very hydrophobic peptide. This hydrophobic region is buried in factor III. But upon phosphorylation of factor III it is turned out. Thus P-FIII binds to Triton X-100 micelles whereas factor III does not. This conformational change caused by phosphorylation could be shown by proton nuclear magnetic resonance methods [Kalbitzer, H.R., Deutscher, J., Hengstenberg, W., & Rösch, P. (1981) Biochemistry 20, 6178-6185], by circular dichroism spectroscopy, and by the Ouchterlony double-diffusion method. Antibodies against FIII do not precipitate P-FIII.

Amino Acid Sequence↗
PubMed1982

HPr proteins of different microorganisms studied by hydrogen-1 high-resolution nuclear magnetic resonance: similarities of structures and mechanisms.

The HPr proteins of Streptococcus lactis, Streptococcus faecalis, Bacillus subtilis, and Escherichia coli were studied by 1H NMR at 360 MHz. The "active-center" histidines of all HPr proteins are characterized by a low pK value between 5.6 and 6.1 and similar spectral parameters. Phosphorylation of the histidyl residues leads to an increase of the pK value of 2-3 units and spectral changes characteristic for N-1 phosphorylation of the histidyl ring. The spectra of the HPr proteins of S. lactis, S. Faecalis, B. subtilis, and Staphylococcus aureus reveal many similarities, whereas the spectrum of the E. coli protein is different with exception of the active-center histidine. The HPr protein of S. lactis is formylated at its terminal amino group.

Bacillus subtilis↗
PubMed1981

Phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus: 1H nuclear magnetic resonance studies on phosphorylated and unphosphorylated factor IIIlac and its interaction with the phosphocarrier protein HPr.

The trimeric phosphocarrier protein factor III specific for galactosides was investigated by 1H NMR spectroscopy. The protomer contains four histidyl residues with acidic pK values in the range 5.6-6.2. One of the histidyl residues, His-B, carries the phosphoryl group. The pK value of His-B increases from 6.0 to 8.6 upon phosphorylation. To determine the position of the phosphoryl group with respect to the nitrogens required the isolation of a peptide T-2 containing the phosphorylated active-center histidine and one of the other histidines. The pK value and the chemical shift of the phosphopeptide clearly indicated the phosphorus to be bound to the N-3 atom of the imidazole ring. The temperature dependence of the factor III spectrum demonstrates multiple conformations which exchange rapidly on the NMR time scale. Titration of factor III with HPr protein showed an upfield shift of the active-center histidine, indicating complex formation between both proteins. Phosphorylation of both proteins abolished the interaction, which is plausible from mechanistic considerations.

Bacterial Proteins↗
PubMed1981

1H nuclear magnetic resonance studies on the structure and mechanism of the HPr protein of Staphylococcus aureus.

1H NMR studies of the phosphocarrier protein HPr and its three nitrotyrosyl derivatives revealed some structural features which may finally lead to an explanation of the mechanism of the phospho-transfer reaction. Titration studies on mononitrated, dinitrated, and trinitrated derivatives--i.e., derivatives with Tyr-56, Tyr-56 and Tyr-37, and Tyr-56, Tyr-37, and Tyr-6 modified--have been performed. The three tyrosyl residues seem to be in positions completely different from each other with respect to their solvent accessibility; Tyr-56 seems to be located near the surface of the protein, Tyr-6 seems to be completely buried, and Tyr-37 takes an intermediate position. Tyr-6 contributes to the core structure of the protein. A resonance at -0.18 ppm could be shown to correspond to a CH3 group of a valine. Nuclear Overhauser experiments revealed its being close to Tyr-6. One of the resonances tentatively assigned to methionine SCH3 groups titrates in the dinitrated derivative with the same pK as nitrotyrosyl residue 37. The titration behavior of the active-center histidyl residue suggests a hydrogen bond to the imidazole ring, possibly from Tyr-56 or Arg-17.

Bacterial Proteins↗
PubMed1981

The staphylococcal phosphoenolpyruvate-dependent phosphotransferase system. Purification and characterisation of the galactoside-specific membrane-component enzyme II.

The galactoside-specific membrane-bound component of the staphylococcal phosphoenolpyruvate-dependent phosphotransferase system, enzyme IIlac, was purified to homogeneity. The purification procedure involved several extractions steps at the particulate state, followed by solubilisation with Triton X-100. Up to this stage the biological activity of enzyme II was preserved. Isolation of the homogeneous protein involved gel filtration of the dodecylsulfate-denatured material. An apparent molecular weight of the polypeptide chain was estimated by dodecylsulfate gel electrophoresis. The 55000-Mr protein is visible in dodecylsulfate gels upon induction of the staphylococcal lac operon as a more intensively stained area. Antibodies against the denatured 55000-Mr protein inhibit the mutant complementation assay of enzyme II offered as membrane fragments. This demonstrates that the 55000-Mr protein and enzyme IIlac are identical. Polarity and the solubility of the protein in detergents are typical for an integral membrane protein.

Amino Acids↗
PubMed1979

The phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus. Complete tyrosine assignments in the 1H nuclear-magnetic-resonance spectrum of the phosphocarrier protein HPr.

Upon nitration of the phosphocarrier protein HPr three nitrated derivatives of the protein were isolated: mononitrated HPr, dinitrated HPr and trinitrated HPr. Tryptic digestion of the derivatives leads to nitrotyrosine-containing peptides which were isolated and characterized by amino acid analysis. This resulted in the determination of the positions of the nitrated tyrosyl residues in the amino acid sequence. In mononitrated HPr only Tyr-56 was modified, in dinitrated HPr both Tyr-56 and Tyr-37 had reacted with the nitrating agent; modification of all three tyrosyl residues in trinitrated HPr required more drastic reaction conditions. The nuclear magnetic resonance spectra of the three derivatives allowed the assignments of the tyrosine resonances as follows: Tyr-A and Tyr-B with pK values of 10.5 and 11.5 were designated Tyr-56 and Tyr-37 whereas Tyr-C, whose protons are not titratable before denaturation of the protein, was assigned to Tyr-6 in the amino acid sequence. The nitration studies, together with the titration behaviour of the three tyrosines, indicate the topology of the tyrosyl residues to be as follows: Tyr-56 is located at the surface, Tyr-37 is slightly buried, Tyr-6 is deeply buried. The nitrotyrosyl derivatives retain their biological activity.

Amino Acids↗
PubMed1977

The phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus. 1. Amino-acid sequence of the phosphocarrier protein HPr.

The primary structure of the histidine-containing phosphocarrier protein HPr of the phosphoenolpyruvate-dependent phosphotransferase system from Staphylococcus aureus was determined by automated Edman degradation. The complete sequence was deduced from the direct analysis of the protein by automated Edman degradation in a liquid-phase sequencer of Edman and from the sequence of tryptic, thermolytic and cyanogen bromide peptides as obtained by automated Edman degradation in a solid-phase sequencer of Laursen. The amino-acid sequence was found to be Met-Glu-Gln-Asn-Ser-Tyr-Val-Ile-Ile-Asp-Glu-Thr-Gly-Ile-His-Ala-Arg-Pro-Ala-Thr-Met-Leu-Val-Gln-Thr-Ala-Ser-Lys-Phe-Asp-Ser-Ile-Asp-Gln-Gly-Gly-Tyr-Asp-Ser-Met-Gln-Leu-Lys-Ser-Leu-Gly-Val-Gly-Lys-Asp-Glu-Glu-Ile-Thr-Ile-Tm-Ser-Ala-Asp-Lys-Lys-Glu-Gly-Leu-Thr-Lys-Met-Ser-Ile-Val. The 70 residues correspond to a molecular weight of 7685. The one histidine involved in the phosphotransfer reaction of this protein was found at position 15 as part of a region of the sequence which has no predictable secondary structure. It is suggested that this protein belongs to the group of male proteins with the active center located on a protrusion rather than a cleft.

Amino Acid Sequence↗
PubMed1977

The phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus. 3. 1H and 31P nuclear-magnetic-resonance studies on the phosphocarrier protein HPr; tyrosine titration and denaturation studies.

The phosphocarrier protein HPr has been investigated by proton nuclear magnetic resonance (NMR) at 270 MHz in order to evaluate structural properties of the whole molecule and its active site. The titration behaviour of the three tyrosines of the HPr protein was analysed by monitoring the chemical shifts of the aromatic proton resonances of these residues as a function of pH. It was found that the HPr protein contains a lot of slowly exchanging NH backbone protons which suggested a relatively rigid secondary structure of the protein molecule itself although it contains no disulfide bridges. The HPr protein shows a sharp reversible denaturation behaviour at alkaline pH values. Between pH 10.8 and 11.1 two C-2 proton resonance peaks for the single histidine residue could be observed together with abrupt changes in the aromatic and aliphatic absorption region of the HPr protein which are due to chemical exchange processes. The NMR spectrum of the HPr protein is only changed a little upon raising the temperature from 14 degrees C to 70 degrees C. At 76 degrees C all resonances in the spectrum broaden and almost disappear. This process is irreversible.

Bacterial Proteins↗