Characterization of carbonic anhydrase from Neisseria gonorrhoeae.

We have investigated the steady state and equilibrium kinetic properties of carbonic anhydrase from Neisseria gonorrhoeae (NGCA). Qualitatively, the enzyme shows the same kinetic behaviour as the well studied human carbonic anhydrase II (HCA II). This is reflected in the similar pH dependencies of the kinetic parameters for CO(2) hydration and the similar behaviour of the kinetics of (18)O exchange between CO(2) and water at chemical equilibrium. The pH profile of the turnover number, k(cat), can be described as a titration curve with an exceptionally high maximal value of 1.7 x 10(6) s(-1) at alkaline pH and a pK(a) of 7.2. At pH 9, k(cat) is buffer dependent in a saturable manner, suggesting a ping-pong mechanism with buffer as the second substrate. The ratio k(cat)/K(m) is dependent on two ionizations with pK(a) values of 6.4 and 8.2. However, an (18)O-exchange assay identified only one ionizable group in the pH profile of k(cat)/K(m) with an apparent pK(a) of 6.5. The results of a kinetic analysis of a His66-->Ala variant of the bacterial enzyme suggest that His66 in NGCA has the same function as a proton shuttle as His64 in HCA II. The kinetic defect in the mutant can partially be overcome by certain buffers, such as imidazole and 1,2-dimethylimidazole. The bacterial enzyme shows similar K(i) values for the inhibitors NCO(-), SCN(-) and N(3)(-) as HCA II, while CN(-) and the sulfonamide ethoxzolamide are considerably weaker inhibitors of the bacterial enzyme than of HCA II. The absorption spectra of the adducts of Co(II)-substituted NGCA with acetazolamide, NCO(-), SCN(-), CN(-) and N(3)(-) resemble the corresponding spectra obtained with human Co(II)-isozymes I and II. Measurements of guanidine hydrochloride (GdnHCl)-induced denaturation reveal a sensitivity of the CO(2) hydration activity to the reducing agent tris(2-carboxyethyl)phosphine (TCEP). However, the A(292)/A(260) ratio was not affected by the presence of TCEP, and a structural transition at 2.8--2.9 M GdnHCl was observed.

Transient kinetics of ligand binding and role of the C-terminus in the dUTPase from equine infectious anemia virus.

Transient kinetics of the equine infectious anemia virus deoxyuridine 5'-triphosphate nucleotide hydrolase were characterized by monitoring the fluorescence of the protein. Rate constants for the association and dissociation of substrate and inhibitors were determined and found to be consistent with a one-step mechanism for substrate binding. A C-terminal part of the enzyme presumed to be flexible was removed by limited trypsinolysis. As a result, the activity of the dUTPase was completely quenched, but the rate constants and fluorescent signal of the truncated enzyme were affected only to a minor degree. We conclude that the flexible C-terminus is not a prerequisite for substrate binding, but indispensable for catalysis.

Kinetic and spectroscopic characterization of native and metal-substituted beta-lactamase from Aeromonas hydrophila AE036.

Two metal ion binding sites are conserved in metallo-beta-lactamase from Aeromonas hydrophila. The ligands of a first zinc ion bound with picomolar dissociation constant were identified by EXAFS spectroscopy as one Cys, two His and one additional N/O donor. Sulfur-to-metal charge transfer bands are observed for all mono- and di-metal species substituted with Cu(II) or Co(II) due to ligation of the single conserved cysteine residue. Binding of a second metal ion results in non-competitive inhibition which might be explained by an alternative kinetic mechanism. A possible partition of metal ions between the two binding sites is discussed.

Preference of Cd(II) and Zn(II) for the two metal sites in Bacillus cereus beta-lactamase II: A perturbed angular correlation of gamma-rays spectroscopic study.

Cd-substituted forms of the Bacillus cereus metallo-beta-lactamases (BCII) were studied by perturbed angular correlation of gamma-rays (PAC) spectroscopy. At very low [Cd]:[apo-beta-lactamase] ratios, two nuclear quadrupole interactions (NQI) were detected. For [Cd]:[apo-beta-lactamase] ratios between 0.8 and 3.0, two new NQIs appear, and the spectra show that up to 2 cadmium ions can be bound per molecule of apoenzyme. These results show the existence of two interacting Cd-binding sites in BCII. The relative populations of the two NQIs found at low [Cd]:[apo-beta-lactamase] ratios yielded a 1:3 ratio for the microscopic dissociation constants of the two different metal sites (when only one cadmium ion is bound). X-ray diffraction data at pH 7.5 demonstrate that also for Zn(II) two binding sites exist, which may be bridged by a solvent molecule. The measured NQIs could be assigned to the site with three histidines as metal ligands (three-His site) and to the site with histidine, cysteine, and aspartic acid as metal ligands (Cys site), respectively, by PAC measurements on the Cys168Ala mutant enzyme. This assignment shows that cadmium ions preferentially bind to the Cys site. This is in contrast to the preference of Zn(II) in the hybrid Zn(II)Cd(II) enzyme, where an analysis of the corresponding PAC spectrum showed that Cd(II) occupied the Cys site, whereby Zn(II) occupied the site with three histidines. The difference between Zn(II) and Cd(II) in affinity for the two sites is combined with the kinetics of hydrolysis of nitrocefin for different metal ion substitutions (Zn(2)E, ZnE, Cd(2)E, CdE, and ZnCdE) to study the function of the two metal ion binding sites.

Endotoxin adsorbent based on immobilized human serum albumin.

Extracorporeal apheresis of endotoxins and pro-inflammatory cytokines is still a therapeutic option in the early hyper-inflammatory phase of gram-negative sepsis. There is therefore ongoing interest in adsorber materials suitable for that kind of clinical application. Here we describe lipopolysaccharide (LPS) and cytokine adsorption characteristics of a new adsorbent based on purified human serum albumin (HSA) covalently linked to macroporous polymer beads (iHSA). Multipoint attachment of HSA to acrylic beads via carboxyl groups of the protein resulted in an increased affinity to LPS. In adsorption experiments (adsorbent/ plasma ratio 1:3) a 70-80% reduction of limulus amoebocyte lysate (LAL) activity from 8.59+/-2.07 EU/ml (mean+/-SD) to 1.82+/-0.77 EU/ml (S. abortus equi; n=40) (p < 0.001) and from 115.13+/-53.76 EU/ml to 17.70+/-11.68 EU/ml (E. coli F583; n=6) (p < 0.01) was achieved. iHSA-purified plasma samples showed a decreased capability of inducing cytokine release from peripheral monocytes. Direct haemoperfusion of LPS pre-stimulated whole blood over iHSA resulted in decreased tumour necrosis factor alpha (TNFalpha) concentrations (30-40% reduction) whereas induced levels of interleukin (IL)-1beta and IL-6 were not affected. Depending on the means of immobilization, iHSA shows higher affinity for LPS than native albumin present in plasma. We demonstrated an efficient removal of LPS from plasma in vitro. Adsorption over immobilized HSA appears to be a simple and effective means of removing LPS and perhaps pro-inflammatory cytokines from the circulation.

Mono- and binuclear Zn2+-beta-lactamase. Role of the conserved cysteine in the catalytic mechanism.

When expressed by pathogenic bacteria, Zn2+-beta-lactamases induce resistance to most beta-lactam antibiotics. A possible strategy to fight these bacteria would be a combined therapy with non-toxic inhibitors of Zn2+-beta-lactamases together with standard antibiotics. For this purpose, it is important to verify that the inhibitor is effective under all clinical conditions. We have investigated the correlation between the number of zinc ions bound to the Zn2+-beta-lactamase from Bacillus cereus and hydrolysis of benzylpenicillin and nitrocefin for the wild type and a mutant where cysteine 168 is replaced by alanine. It is shown that both the mono-Zn2+ (mononuclear) and di-Zn2+ (binuclear) Zn2+-beta-lactamases are catalytically active but with different kinetic properties. The mono-Zn2+-beta-lactamase requires the conserved cysteine residue for hydrolysis of the beta-lactam ring in contrast to the binuclear enzyme where the cysteine residue is not essential. Substrate affinity is not significantly affected by the mutation for the mononuclear enzyme but is decreased for the binuclear enzyme. These results were derived from kinetic studies on two wild types and the mutant enzyme with benzylpenicillin and nitrocefin as substrates. Thus, targeting drug design to modify this residue might represent an efficient strategy, the more so if it also interferes with the formation of the binuclear enzyme.

Mono- and binuclear Zn-beta-lactamase from Bacteroides fragilis: catalytic and structural roles of the zinc ions.

The Bacteroides fragilis Zn-beta-lactamase is active with a mono- and a binuclear zinc site. The apoenzyme produced by removal of both Zn ions does not recover full activity upon readdition of Zn2+ in contrast to an active mono-Zn form prepared at pH 6.0. Differences in k(cat) values observed are substrate-dependent implying distinct mechanisms for the mono- and binuclear species. The substrate profile of a Zn,Cd hybrid obtained by selective exchange of one zinc ion is different from that of the Zn2 enzyme with a remarkable 15-fold increased activity with cefoxitin as substrate.

Ric c 1 and Ric c 3, the allergenic 2S albumin storage proteins of Ricinus communis: complete primary structures and phylogenetic relationships.

The 2S albumin storage protein of Ricinus communis consists of the two heterodimeric proteins Ric c 1 and Ric c 3 each of which is composed of a small and a large subunit linked together by disulphide bridges. The complete primary structures of both heterodimeric proteins were determined by enzymatic degradation and automated Edman degradation. The sequences of all four chains correspond to the known cDNA sequence of the gene of a presumed precursor molecule and to the previously determined partial sequences for Ric c 1 and Ric c 3. In addition, few differences in amino acid positions were found which seem to be related to different varieties of R. communis. Sequence comparisons with 2S albumin from other plant genera revealed high degrees of homology and support the view of a common genetic origin of this protein family. Ric c 1 and Ric c 3 which have 11,212 and 12,032 daltons, respectively, share a similar molecular size, biological function and allergenicity with the 2S albumins from Brassica juncea (Braj 1E) and Sinapis alba L (Sin a 1). Ric c 1 and Ric c 3 may be classified as isoallergens if, additionally, the high degree of similarity in the position of polar residues is taken into account.

Zn(II) dependence of the Aeromonas hydrophila AE036 metallo-beta-lactamase activity and stability.

Two Zn2+ binding sites were found in the Aeromonas hydrophila AE036 metallo-beta-lactamase. The affinity of the first binding site for Zn2+ ions is so high that the dissociation constant could not be determined, but it is significantly lower than 20 nM. The mono-Zn2+ form of the enzyme exhibits a maximum activity against its carbapenem substrates. The presence of a Zn2+ ion in the second lower affinity binding site results in a loss of enzymatic activity with a Ki value of 46 microM at pH 6.5. The kinetic analysis is in agreement with a noncompetitive inhibition mechanism. The Zn content of the A. hydrophila enzyme is also strongly pH-dependent. With an external Zn2+ ion concentration of 0.4 microM, occupancy of the higher affinity site by metal ions is lower than 10% at pH 5 and 10. The affinity for the second binding site seems to increase from pH 6 to 7.5. Fluorescence emission and circular dichroism spectra revealed slight conformational changes upon titration of the apoenzyme by Zn2+ ions, resulting in the successive saturation of the first and second binding sites. Differential scanning calorimetry transitions and intrinsic fluorescence emission spectra in the presence of increasing concentrations of urea demonstrate that the catalytic zinc strongly stabilizes the conformation of the enzyme whereas the di-Zn enzyme is even more resistant to thermal and urea denaturation than the mono-Zn enzyme. The Zn2+ dependency of the activity of this metallo-beta-lactamase thus appears to be very different from that of the homologous Bacteroides fragilis enzyme for which the presence of two Zn2+ ions per molecule of protein appears to result in maximum activity.

Cloning and expression of the Epstein-Barr virus-encoded dUTPase: patients with acute, reactivated or chronic virus infection develop antibodies against the enzyme.

The gene encoding the Epstein-Barr virus (EBV)-specific dUTPase was amplified from virus DNA by PCR. The active enzyme was expressed in Escherichia coli and in insect cells as a non-fusion protein. The protein from E. coli specifically converted dUTP to dUMP and did not react with other dNTPs or NTPs. Preliminary experiments yielded a Km value of about 0.8 microM for dUTP. MAbs against the dUTPase reacted with a protein of approximately 31 kDa in 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-stimulated B cells harbouring either type 1 or type 2 EBV. The protein was found in untreated cells at low levels, whereas induction of the lytic replication cycle by TPA treatment or by providing the immediate early transactivator BZLF1 in trans resulted in increased expression. We demonstrated that the virus dUTPase isolated from EBV-infected cells is a phosphoprotein. The protein expressed in insect cells was used to test for the presence of specific antibodies in sera from normal, healthy carriers and from patients with various diseases. While the sera of EBV-negative individuals (0/3) or healthy carriers (0/33) did not contain detectable levels of antibodies, patients with mononucleosis (5/18), chronic EBV infection (2/7), EBV reactivation (7/20) and human immunodeficiency virus infection (5/24) showed elevated antibody titres against the enzyme. This indicated that the dUTPase is expressed during EBV replication and reactivation. The enzyme might therefore be a potential target for drug therapy under conditions of active DNA replication.

Histone H1 suppresses tumor growth of leukemia cells in vitro, ex vivo and in an animal model suggesting extracellular functions of histones.

Purified histone H1 exerts growth inhibition of leukemia cells independent of lineage, stage, and maturation. At 200 micrograms/ml, H1 proved cytotoxic in 19 of 21 of the tested leukemia-derived cell lines and for 11 of 16 of the fresh tumor samples from leukemia patients. In all cases, normal peripheral blood mononuclear cells and bone marrow cells remained unaffected. Multicellular spheroids from the Burkitt's lymphoma cell line IM-9 were growth arrested at 500 micrograms H1/ml. The clonogenic growth of the Burkitt's lymphoma cell line Daudi was arrested at 160 micrograms H1/ml. Synthetic H1-peptides as well as peptides and proteins with biochemical properties similar to H1 had no inhibitory growth effect at equimolar concentrations. Furthermore, 250 micrograms H1 injected into a Burkitt's lymphoma (Daudi), xenotransplanted into nude mice, arrested tumor growth. As shown by electron microscopy and flow cytometry, incubation of leukemia cells with H1 resulted in severe plasma membrane damage and ultimately cytolysis. This report characterizes a 33-kd protein that binds H1 and is responsible for the cell death via destruction of the cell membrane integrity. New extranuclear functions of histones are presented.

The protein conformation of Cd-substituted horse liver alcohol dehydrogenase and its metal-site coordination geometry in binary and ternary inhibitor complexes.

The coordination geometry of the metal at the active site in Cd-substituted horse liver alcohol dehydrogenase (LADH) has been investigated for the binary complexes of LADH with imidazole, isobutyramide, decanoic acid and Cl-, and for the ternary complexes of LADH with NADH and imidazole, NADH and isobutyramide, NAD+ and decanoic acid and NAD+ and Cl-, by using the method of perturbed angular correlation of gamma-rays (PAC). The spectral results are consistent with a flexible structure around the metal for the binary complexes with inhibitors. For ternary complexes, however, a rigid structure is observed. An exception is the ternary complex between LADH, NADH and imidazole, in which the metal site is still flexible. Comparing with available structures determined by X-ray crystallography, we found a correlation between open structures and flexible metal sites, and between closed structures and rigid metal sites. This indicates that the PAC technique can be applied to distinguish the two conformations in solution. The spectral parameters, omega(o) and eta, of the experiments, except for the complexes with imidazole, fall into two groups: one with low omega(o) and one with high omega(o) (eta is relatively constant in all experiments). In this work it is clarified that the low omega(o) values are connected with the presence of a negatively charged solvent ligand. Using an angular-overlap approach to interpret the results, the low omega(o) values are found to be compatible with a coordination geometry where the S-Cd-S (Cys174 and Cys46 coordinate to the metal) angle is about 110 degrees as suggested in [Hemmingsen, L., Bauer, R., Danielsen, E., Bjerrum. M. J., Zeppezauer, M., Adolph, H. W., Formicka, G. & Cedergren-Zeppezauer, E. (1995) Biochemistry 34, 7145-7153], whereas high omega(o) values are compatible with an S-Cd-S angle of 130 degrees. The presence of a negatively charged metal ligand, therefore, might trigger the movement of the sulfur of Cys174. As it is believed that alcohols coordinate to the metal as alcoholate ions this could be important for catalysis.

[Blood pressure adaptation and hormone regulation in Parkinson patients following orthostasis]. / Blutdruckanpassung und hormonelle Regulation bei Parkinson-Patienten nach Orthostase.

OBJECTIVE: To determine whether abnormal orthostatic regulation commonly occurs in patients with Parkinson's disease (PD) and is caused by an abnormal catecholamine release (especially of noradrenaline). PATIENTS AND METHODS: Blood pressure and catecholamine concentration were measured after 10-min recumbency and 1, 3, 5, 7 and 9 min after standing up in 46 patients with PD (15 women, 31 men: mean age 64.3 +/- 11.7 years). Results were compared with those in a group of 27 healthy controls (eight women, 19 men; mean age 60.7 +/- 10.8 years). RESULTS: Among PD patients there were marked hypotonic circulatory reactions, associated with decreased catecholamine concentration and heart rate adaptation; an abnormal orthostatic regulation occurred in 30 patients. In 22 patients noradrenaline concentration changed by less than 200 pg/ml in the 9th min after standing up, while a decreased hormonal release was noted in only four of the controls, all of whom had normal orthostatic regulation. In PD patients the lowest adrenaline rise was always associated with an extremely high fall in blood pressure. Changes in mean blood pressure correlated positively with the rise in noradrenaline. There was no demonstrable correlation between medication for PD, age of patient and the duration or severity of the disease. CONCLUSION: Abnormal orthostatic regulation occurs more frequently in PD than in healthy persons and is associated with changes in noradrenaline release. This suggests multisystem degeneration as a cause of PD.

Magnetic and optical properties of copper-substituted alcohol dehydrogenase: a bisthiolate copper (II) complex.

Replacement of the catalytic Zn(II) in horse liver alcohol dehydrogenase (HLADH) with copper produces a mononuclear Cu(II) chromophore with a ligand set consisting of two cysteine sulphurs, one histidine nitrogen plus one further atom. The fourth ligand to the metal ion and the conformation of the protein may be altered by addition of exogenous ligands and/or the cofactor NADH. Absorbance, CD, low-temperature magnetic CD (MCD) and EPR spectra are presented of copper-substituted HLADH samples in both 'open' and 'closed' conformations and in the presence and absence of the exogenous ligands pyrazole and DMSO. The EPR spectra indicate a strong, predominantly axial field about the copper(II) ion with high copper-thiol (cysteine) covalence. The optical and MCD spectra are interpreted in terms of four d-d transitions to low energy, also reflecting the axial ligand field, and four charge-transfer transitions to copper(II) between 30000 and 16000 cm-1 arising from the two cysteine sulphur atoms which give two pairs of oppositely signed MCD C-terms. These transitions are polarized mainly in the axial plane defined by Cys-46, Cys-174 and His-67. The binary complex formed with pyrazole displays quite different EPR and optical spectra which can be understood in terms of a rotation of the copper hole-orbital away from the axial plane thus decreasing sharply the copper-thiol covalence. The magneto-optical spectra in the presence and absence of DMSO are indistinguishable.

Specific derivatization of the active site tyrosine in dUTPase perturbs ligand binding to the active site.

Selective modification of one (of three) tyrosine residue per enzyme monomer leads to inactivation of dUTPase of the retrovirus equine infectious anemia virus (EIAV). The substrate dUMP and the cofactor Mg2+ protect against inactivation and modification, in agreement with the study on E. coli dUTPase (Vertessy et al. (1994) Biochim. Biophys. Acta 1205, 146-150). Amino acid analyses of nitrated dUTPases confirmed Tyr-selectivity of modification. The nitrated residue in E. coli dUTPase was identified as the evolutionary highly conserved Tyr-93. The modifiable residue is shown to be the only Tyr exposed in both E. coli and EIAV dUTPases. As a consequence of Tyr-93 derivatization, the Mg2+-dependent interaction between the substrate-analogue dUDP and E. coli dUTPase becomes impaired as shown by circular dichroism spectroscopy, here presented as a tool for monitoring ligand binding to the active site.

Separation and characterisation of bovine histone H1 subtypes by combined ion-exchange and reversed-phase chromatography and mass spectrometry.

In order to separate and identify histone H1 subtypes from calf thymus we used both electrospray mass spectrometry (ES-MS) and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) after a three-step chromatographic procedure consisting of reversed-phase high-performance liquid chromatography (RP-HPLC), size-exclusion chromatography (SEC) and ion-exchange chromatography (IEC). Under the RP-HPLC conditions described, we obtained two baseline-separated H1-fractions which were characterised by MALDI-TOF-MS. The determined masses ranged from 22,850 to 22,590 for the first fraction and from 22,070 to 21,250 for the second fraction. Further, it was shown that the first fraction contained at least four and the second one at least five subtypes of the histone class H1. Four homogeneous pure H1 subtypes were obtained by a combination of IEC followed by SEC and RP-HPLC. The molecular masses of these four subtypes determined by ES-MS were 22,606, 22,761, 21,347 and 21,263. We obtained six additional molecular masses of histone H1 subtypes from three heterogeneous fractions, namely 22,066, 21,802, 20,586 and 19,817 by ES-MS and 22,800 and 22,675 by MALDI-TOF-MS. The retention times of these fractions and the molecular masses were in agreement with the data obtained from RP-HPLC fractions by MALDI-TOF-MS.

Cd-substituted horse liver alcohol dehydrogenase: catalytic site metal coordination geometry and protein conformation.

The coordination geometry of the catalytic site in Cd-substituted horse liver alcohol dehydrogenase (LADH) has been investigated as a function of pH using the method of perturbed angular correlation of gamma-rays (PAC). LADH in solution fully loaded with cadmium, including radioactive 111mCd in the catalytic site [Cd2(111mCd)Cd2LADH], was studied over the pH range 7.9-11.5. Analysis of the PAC spectra showed the ionization of a group with pKa of 11. This pKa value is about 2 pH units higher than that of native zinc-containing LADH. A pKa of 9.6 was found for the binary complex of Cd2(111mCd)Cd2LADH with NAD+. This value is also about 2 pH units higher than that of the binary complex of native zinc-containing enzyme and NAD+. No pH dependency was detected for the binary complex of Cd2(111mCd)Cd2LADH with NADH within the pH range measured (pH 8.3-11.5). Assuming that metal-coordinated water is the ionizing group [Kvassman, J., & Pettersson, G. (1979) Eur. J. Biochem. 100, 115-123], we conclude that the larger ionic radius of Cd(II) relative to Zn(II) in the catalytic site causes the elevated pKa values of metal-bound water. Interpretation of nuclear quadrupole interaction (NQI) parameters derived from PAC spectra is based on the use of the angular overlap model, using the coordinates for the catalytic zinc site from the 1.8 A resolution crystal structure of the ternary complex between LADH, NADH, and dimethyl sulfoxide as a model.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical phosphorylation of the peptides GGXA (X = S, T, Y): an evaluation of different chemical approaches.

An evaluation was made of the two methods most commonly used for phosphorylation of hydroxyamino acids in peptides, i.e. the tetrazole-catalysed phosphitylation by di-tert-butyl-N,N-diethylphosphoramidite followed by oxidation and the phosphorylation by dibenzylphosphochloridate. As model system the sequence GGXA (X = S, T, Y) was used which represents a random-coil sequence avoiding the influence on the reaction kinetics of secondary structure formation. In the case of serine- and threonine-containing peptides, both synthetic methods gave comparable yields of the desired phosphopeptides. The phosphorylation of tyrosine was achieved more favorably via the phosphoramidite method. However, phosphotyrosine peptides are most easily obtained by peptide synthesis using Fmoc-Tyr(PO3Me2)OH as building block. The dibenzylphosphochloridate method yields the expected phosphopeptides as the only peptide derivative and in addition, a great number of unidentified by-products which can be removed by ion-exchange chromatography. The phosphoramidite method consistently resulted in three peptide derivatives, i.e. the desired phosphopeptide, the phosphitylated peptide and a bridged derivative with two GGXA fragments linked through a phosphodiester bridge. The derivatives were characterised by RP and ion-exchange chromatography, 31P- and 1H-NMR spectroscopy, and ion-spray and electrospray mass spectrometry. Interestingly, even these mild ionisation techniques resulted in partial fragmentation. The observed fragmentation pathways seem to be a diagnostic tool for the identification of phosphorylation sites in peptides. Both the phosphorylated serine and threonine peptide lost phosphoric acid (98 mass units), the tyrosine peptide lost phenyl phosphate (174 mass units).(ABSTRACT TRUNCATED AT 250 WORDS)

1H and 31P NMR spectroscopy of phosphorylated model peptides.

The model peptides glycylglycyltyrosylalanine (Gly-Gly-Tyr-Ala), glycylglycylthreonylalanine (Gly-Gly-Thr-Ala) and glycylglycylserylalanine (Gly-Gly-Ser-Ala) were phosphorylated at the hydroxyl groups of their tyrosyl, threonyl and seryl residues, respectively, and characterized by 31P and 1H NMR spectroscopy. The pKa-value of the phosphoryl group in the tyrosine-containing peptide determined from the pH dependence of chemical shifts is 5.9, the 31P chemical shifts at low pH (4.0) and high pH (8.0) are -3.8 and 0.2 ppm, respectively. Phosphorylation also leads to significant shifts of the 1H NMR resonances of the tyrosine residue; the amide resonance is shifted -0.02 ppm, the H alpha resonance 0.06 ppm, the H beta resonances 0.10 and -0.04 ppm, the H delta resonances 0.02 ppm and the H epsilon resonances 0.26 ppm. The pKa-value of the phosphoryl group in the threonine peptide determined from the pH dependence of chemical shifts is 6.1; the 31P chemical shifts at low pH (4.0) and high pH (8.0) are -0.1 and 4.8 ppm, respectively. The corresponding values for the serine peptide are 6.1 (pKa), 0.6 ppm and 4.9 ppm. Phosphorylation also leads to significant shifts of the 1H NMR resonances of the threonine and serine residues. In the threonine residue the amide resonance is shifted 0.25 ppm, the H alpha-resonance -0.43 ppm, the H beta-resonance 0.03 ppm and the H gamma-resonance 0.09 ppm. In the serine residue the amide resonance is shifted 0.21 ppm, the H alpha-resonance -0.17 ppm, and the H beta-resonances 0.17 ppm.