Vanadium(V) complexes of alpha-hydroxycarboxylic acids in aqueous solution.

51V NMR and IR spectroscopic studies of the complexes formed between vanadate and the alpha-hydroxylic acid ligands, (S)-2-hydroxypropanoic acid (L-(+)-lactic acid), 2-hydroxy-2-methylpropanoic acid, and 2-ethyl-2-hydroxybutanoic acid were carried out for aqueous 1 M ionic strength (NaCl) solutions. Three major products in V to L stoichiometries of 1:1, 2:2, and 3:2 were identified from vanadate and ligand concentration studies, while a pH variation study allowed charge states to be determined. At pH 7.06, the formation constants for the predominant reactions were (26 +/- 1) M (-1), (V + L <= => VL); (6.8 +/- 0.4) x 10(3) M(-1), (2VL <= => V(2)L(2)); and (3.5 +/- 0.3) x 10(3) M(-1), (V(2)L(2) + V <= => V(3)L(2)). Dissolution studies of various crystalline products were carried out for aqueous, nonaqueous, and mixed solvent systems. These studies combined with information available from X-ray structural studies provided a basis for the assignment of solution state structures. Pentacoordinate vanadium in a trigonal-bipyramidal geometry was proposed for the both the 1:1 and 2:2 complexes when in aqueous solution. Observed changes in (51)V chemical shift patterns were consistent with a cis fusion in octahedral coordination for the central vanadium of the 3:2 complex, while the remaining vanadiums retained a pentacoordinate geometry.

Vanadium(V) complexes in enzyme systems: aqueous chemistry, inhibition and molecular modeling in inhibitor design.

Vanadate in aqueous solution is known to influence a number of enzyme-catalyzed reactions. Such effects are well known to carry over to living systems where numerous responses to the influence of vanadium have been well-documented; perhaps the most studied being the insulin-mimetic effect. Studies of the aqueous chemistry of vanadate provide an insight into the mechanisms by which vanadate affects enzyme systems and suggests methods for the elucidation of specific types of responses. Studies of the corresponding enzymes provide complementary information that suggests model vanadate systems be studied and provides clues as to functional groups that might be utilized in the development of selective enzyme inhibition. The insulin-mimetic effect is thought by many workers to originate in the effectiveness of vanadium as an inhibitor of protein tyrosine phosphatase (PTPase) activity. One, or more PTPases regulate the phosphotyrosine levels of the insulin receptor kinase domain. Appropriate ligands allow modification of the reactivity and function of vanadate. For instance, although the complex, ((CH(3))(2)NO)(2)V(O)OH, is not quite as good an inhibitor of PTPase activity as is vanadate, it is much more effective in cell cultures for increasing glucose transport and glycogen synthesis. Studies of the chemistry of this complex provide an explanation of the efficacy of this compound as a PTPase inhibitor that is supported by computer modeling studies. Computer calculations using X-ray data of known PTPases as a basis for homology modeling then suggests functionality that needs to be addressed in developing selective PTPase inhibitors.

Hydroxamido vanadates: aqueous chemistry and function in protein tyrosine phosphatases and cell cultures.

The protein tyrosine phosphatases (PTPases) are a group of regulatory enzymes that are critically important to a wide variety of cellular functions. A number of these PTPases have significant potential as targets for therapeutic intervention, for instance, in diabetes and autoimmune disease treatment. The hydroxylamine complex, bis(N,N-dimethylhydroxamido)hydroxooxovanadate (DMHAV), is an excellent inhibitor of the two PTPases, protein tyrosine phosphatase 1B (PTP1B) and leucocyte common antigen related phosphatase (LAR). However, because of the similarity of the active site architecture within the group of known PTPases, DMHAV is probably an effective inhibitor of most PTPases. Information gleaned from studies of the mechanism of inhibition of PTPases by peptide-derived inhibitors, together with information from comparative protein modelling and studies of the aqueous chemistry of DMHAV, has provided insights for the development of selective PTPase inhibitors. In cell cultures, DMHAV is effective in increasing phosphotyrosine levels on the insulin receptor and greatly facilitates glucose transport and glycogen synthesis. Selective PTPase inhibitors that are developed from the basis of the hydroxylamine motif may lead to effective vanadate-based complexes that have potential as therapeutic agents.

The phosphatase domains of LAR, CD45, and PTP1B: structural correlations with peptide-based inhibitors.

PTP1B is a cytosolic protein tyrosine phosphatase that is a regulator of the kinase activity of the insulin receptor; the two protein tyrosine phosphatases LAR and CD45 are receptor type phosphatases crucially important to cell function. LAR also is involved in regulation of the insulin receptor while CD45 is critical for T-cell activation. Although LAR and CD45 are both transmembrane phosphatases, these enzymes manifest their phosphatase activity through a catalytic cytosolic domain. We have utilized X-ray coordinates of related phosphatases (RPTPalpha and RPTmu) and comparative protein modeling to obtain molecular models of the D1 catalytic domains of CD45 and LAR. The models were tested using established protocols and found to be comparable to low resolution X-ray structures. The structure obtained for LAR was compared with the recently reported X-ray structure. Both the CD45-D1 and LAR-D1 structures were then compared to and contrasted with PTP1B. The active site of pockets of the three enzymes were found to be very uniform in structure and charge distribution. Also, the gross surface topology around the active site was found to be somewhat similar for the 3 phosphatases. However, there were significant differences in surface topology, and, more importantly, large changes in surface charge distribution. The differences between the surface features of these enzymes provide an explanation for the selectivity of inhibition by a number of peptides.

Structure, modelling, and molecular dynamics studies of the inhibition of protein tyrosine phosphatase 1B by sulfotyrosine peptides.

The protein tyrosine phosphatases comprise a class of enzymes that are crucial for the regulation of a number of cellular processes. Because of this, they are attracting increasing attention, not only as legitimate therapeutic targets, but also because of their relationship to many fundamental cellular processes. Certain sulfotyrosine peptides derived from casein are known to be good inhibitors of the protein tyrosine phosphatase, PTP1B. In this study, NMR transfer nuclear Overhauser effect studies have been used to ascertain the bound-state conformation adopted by the 12-amino acid residue casein-derived peptide, CAS200 (NANEEE(sY)SIGSA) and N-terminal truncated forms of this peptide, CAS203 and CAS205. Each of the peptides were found to bind in an extended beta-strand conformation. Extensive molecular modelling and molecular dynamics simulations of the PTP1B/peptide complexes, in a fully hydrated model, allowed a detailed description of the potential sources of the binding interactions to be developed. In agreement with the NMR studies, the modelling provided a picture of binding of CAS200 in which only the central (E203-I208) residues contributed significantly to the binding while the 3 N-terminal and 3 C-terminal residues were quite fluxional. Critical cationic surface residues, lying near to, but outside the active site pocket were the source of strong stabilizing forces that complemented the stabilizing interactions of the active site pocket. Electrostatic, hydrophobic, and hydrogen bonding interactions, in a residue specific manner, were all found to make significant contributions to the binding of these inhibitors.

Bis(N,N-dimethylhydroxamido)hydroxooxovanadate inhibition of protein tyrosine phosphatase activity in intact cells: comparison with vanadate.

We have shown previously that bis(N,N-dimethylhydroxamido)hydroxooxovanadate (DMHV) is an excellent reversible inhibitor of protein tyrosine phosphatase (PTP) in vitro. DMHV does not carry a charge under physiological pH conditions and is anticipated to permeate cell membranes more easily than vanadate. In the present study, the efficacy of DMHV as a PTP inhibitor in intact cells was compared with that of vanadate by measuring phosphotyrosine levels in various cells treated with these compounds. DMHV was more effective in increasing both the phosphotyrosine levels of various proteins in 3T3L1 fibroblasts and the level of insulin-receptor phosphorylation in CHO cells overexpressing the human insulin receptor. DMHV was about 10- to 20-fold more effective than vanadate in increasing glucose transport and glycogen synthesis in 3T3L1 adipocytes. DMHV, unlike vanadate, also inhibited PTP in Jurkat cells. The implications of these observations are discussed.

Vanadate inhibition of protein tyrosine phosphatases in Jurkat cells: modulation by redox state.

Vanadate is a potent reversible inhibitor of protein tyrosine phosphatases (PTP) in vitro. Vanadate has been shown to increase the phosphotyrosine levels in some cell types whereas in others, like the Jurkat T-lymphoma, vanadate has no effect. The reason for the apparent lack of effect of vanadate in Jurkat cells was investigated in this study. Alteration of the redox state of these cells by reducing the glutathione level with 1-chloro-2,4-dinitrobenzene (DnpCl) had no effect on phosphotyrosine levels. However, the cells became sensitive to vanadate, as measured by an increase in phosphotyrosine levels on a wide range of proteins including the MAP kinases. The increase in phosphotyrosine levels most likely results from inhibition of cellular PTP and suggests that protein tyrosine kinases are constitutively active in cells, resulting in a dynamic phosphorylation-dephosphorylation cycle. The mode of inhibition of PTP by vanadate was investigated by measuring the PTP activity of Jurkat membranes isolated after treatment of cells with vanadate and DnpCl. In contrast to the reversible inhibition of PTP in vitro, the effect of vanadate in the presence of DnpCl was irreversible, raising the possibility that it is peroxovanadate formed in situ that is responsible for the inhibition of PTP in intact cells.

Nuclear magnetic resonance and restrained molecular dynamics studies of the interaction of an epidermal growth factor-derived peptide with protein tyrosine phosphatase 1B.

The epidermal growth factor-derived (EGFR988) fluorophosphonate peptide, DADE(F2Pmp)L, is a potent (30 pM) inhibitor of the protein tyrosine phosphatase PTP1B. Nuclear magnetic resonance (NMR) transferred nuclear Overhauser effect (nOe) experiments have been used to determine the conformation of DADE(F2Pmp)L while bound in the active site of PTP1B. When bound, the peptide adopts an extended beta-strand conformation. Molecular modeling and molecular dynamics simulations allowed the elucidation of the sources of many of the interactions leading to binding of this inhibitor. Electrostatic, hydrophobic, and hydrogen-bonding interactions were all found to contribute significantly to its binding. However, despite the overall tight binding of this inhibitor, the N-terminal and adjacent residue of the peptide were virtually unrestrained in their motion. The major contributions to binding arose from hydrophobic interactions at the leucine and at the aromatic center, hydrogen bonding to the pro-R fluorine of the fluorophosphonomethyl group, and electrostatic interactions involving the carboxylate functionalities of the aspartate and glutamate residues. These latter two residues were found to form tight contacts with surface recognition elements (arginine and lysine) situated near the active-site cleft.

Structure of the membrane binding domain of CTP:phosphocholine cytidylyltransferase.

It has been proposed that the domain of the regulatory enzyme, CTP:phosphocholine cytidylyltransferase, which mediates reversible binding of the enzyme to membranes, is an amphipathic alpha-helix of approximately 60 amino acid residues and that this domain is adjacent to the putative active site domain of this enzyme. Circular dichroism indicated that the secondary structures of two overlapping peptides spanning this region were predominantly alpha-helical in the presence of PG vesicles or sodium dodecyl sulfate micelles. Interproton distances were obtained from two-dimensional NMR spectroscopic measurements to solve the structures of these two peptides. The C-terminal 22 amino acid peptide segment (corresponding to Val267-Ser288) was a well-defined alpha-helix over its length. The N-terminal 33-mer (corresponding to Asn236-Glu268) was composed of an alpha-helix from Glu243 to Lys266, a well-structured bend of about 50 degrees at Tyr240-His241-Leu242, and an N-terminal four-residue helix. It is proposed that the three residues involved in generating the bend act as the hinge between the catalytic and regulatory domains. The nonpolar faces of the 33-mer and 22-mer were interrupted by Ser260, Ser271, and Ser282. These residues may serve to limit the hydrophobicity and facilitate reversible and lipid-selective membrane binding. The hydrophobic faces of the helices were flanked by a set of basic amino acid residues on one side and basic amino acid residues interspersed with glutamates on the other. The disposition of these side chains gives clues to the basis for the specificities of these peptides for anionic surfaces.

Structural studies of a peptide activator of human lecithin-cholesterol acyltransferase.

The synthetic lipid-associating peptide, LAP-20 (VSSLLSSLKEYWSSLKESFS), activates lecithin-cholesterol acyltransferase (LCAT) despite its lack of sequence homology to apolipoprotein A-I, the primary in vivo activator of LCAT. Using SDS and dodecylphosphocholine (DPC) to model the lipoprotein environment, the structural features responsible for LAP-20's ability to activate LCAT were studied by optical and two-dimensional 1H NMR spectroscopy. A large blue shift in the intrinsic fluorescence of LAP-20 with the addition of detergent suggested that the peptide formed a complex with the micelles. Analysis of the CD data shows that LAP-20 lacks well defined structure in aqueous solution but adopts helical, ordered conformations upon the addition of SDS or DPC. The helical nature of the peptides in the presence of both lipids was confirmed by upfield H alpha NMR secondary shifts relative to random coil values. Average structures for both peptides in aqueous solutions containing SDS and DPC were generated using distance geometry methods from 329 (SDS) and 309 (DPC) nuclear Overhauser effect-based distance restraints. The backbone (N, Calpha, C=O) RMSD from the average structure of an ensemble of 17 out of 20 calculated structures was 0.41 +/- 0.15 Angstrom for LAP-20 in SDS and 0.41 +/- 0.12 A for an ensemble of 20 out of 20 calculated structures for LAP-20 in DPC. In the presence of SDS, the distance geometry and simulated annealing calculations show that LAP-20 adopts a well defined class A amphipathic helix with distinct hydrophobic and hydrophilic faces. A similar structure was obtained for LAP-20 in the presence of DPC, suggesting that both detergents may be used interchangeably to model the lipoprotein environment. Conformational features of the calculated structures for LAP-20 are discussed relative to models for apolipoprotein A-I activation of LCAT.

Inhibition of phosphate-metabolizing enzymes by oxovanadium(V) complexes.

The chemical similarities between vanadate and phosphate combined with the ability of vanadate to readily undergo changes in coordination geometry allows this ion to strongly influence the function of a large variety of phosphate-metabolizing enzymes. As transition state analogs, spontaneously formed vanadate complexes are potent inhibitors of a number of enzymes, including some ribonucleases, mutases, and phosphatases. In addition, vanadate is an effective inhibitor of many ATPases, kinases, lyases, and synthases. Vanadate oligomers tend to be weaker inhibitors than vanadate but do influence the function of dehydrogenases, mutases, aldolases, kinases, and others. Of the oligomers, decavanadate is unique in that it seems to bind only in polyphosphate binding domains. Peroxovanadate has not yet been well studied but it seems to inhibit enzymes that do not utilize a pentacoordinate vanadate in the catalysis cycle. Additional detailed studies of vanadate-initiated inhibition of enzymes will expand our understanding of the various mechanisms of action of vanadate and its derivatives that have been briefly described here and will doubtless provide insight into other functions of this unique material.

1H and 51V NMR studies of the interaction of vanadate and 2-vanadio-3-phosphoglycerate with phosphoglycerate mutase.

The formation of complexes of vanadate with 2-phosphoglycerate and 3-phosphoglycerate have been studied using 51V nuclear magnetic resonance spectroscopy. Signals attributed to two 2,3-diphosphoglycerate analogues, 2-vanadio-3-phosphoglycerate and 2-phospho-3-vanadioglycerate, were detected but were not fully resolved from signals of inorganic vanadate and the anhydride formed between vanadate and the phosphate ester moieties of the individual phosphoglycerates. Equilibrium constants for formation of the two 2,3-bisphosphate analogues were estimated as 2.5 M-1 for 2-vanadio-3-phosphoglycerate and 0.2 M-1 for 2-phospho-3-vanadioglycerate. The results of the binding study are fully consistent with non-cooperativity in the binding of vanadiophosphoglycerate to the two active sites of phosphoglycerate mutase (PGM). 2-Vanadio-3-phosphoglycerate was found to bind to the dephospho form of phosphoglycerate mutase with a dissociation constant of about 1 x 10(-11) M at pH 7 and 7 x 10(-11) M at pH 8. Three signals attributed to histidine residues were observed in the 1H NMR spectrum of phosphoglycerate mutase. Two of these signals and also an additional signal, tentatively attributed to a tryptophan, underwent a chemical shift change when the vanadiophosphoglycerate complex was bound to the enzyme. The results obtained here are in accord with these vanadate-phosphoglycerate complexes being much more potent inhibitors of phosphoglycerate mutase than either monomeric or dimeric vanadate. The dissociation constant of 10(-11) M for 2-vanadio-3-phosphoglycerate is about 4 orders of magnitude smaller than the Km for PGM, a result in accordance with the vanadiophosphoglycerates being transition state analogues for the phosphorylation of PGM by 2,3-diphosphoglycerate.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution and half-life for retention of vanadium in the organs of normal and diabetic rats orally fed vanadium(IV) and vanadium(V).

The concentration of vanadium in organs of diabetic rats that had been fed vanadium, either as V(IV) or V(V), in their drinking water has been determined. The kidney was found to have the highest concentration, about 185 nmol/g wet tissue. This averages about three times higher than for the liver or spleen, for which concentrations were comparable. The lung, blood plasma, and blood cells tended to have the lowest accumulations of vanadium. A time-course study indicated that the half-life for elimination of vanadium from the bodies of vanadium-fed rats is about 12 d.

2,3-diphosphoglycerate phosphatase activity of phosphoglycerate mutase: stimulation by vanadate and phosphate.

The binding of inorganic vanadate (Vi) to rabbit muscle phosphoglycerate mutase (PGM), studied by using 51V nuclear magnetic resonance spectroscopy, shows a sigmoidal dependence on vanadate concentration with a stoichiometry of four vanadium atoms per PGM molecule at saturating [Vi]. The data are consistent with binding of one divanadate ion to each of the two subunits of PGM in a noncooperative manner with an intrinsic dissociation constant of 4 X 10(-6) M. The relevance of this result to other studies which have shown that the Vi-stimulated 2,3-diphosphoglycerate (2,3-DPG) phosphatase activity of PGM has a sigmoidal dependence on [Vi] with a Hill coefficient of 2.0 is discussed. At pH 7.0, inorganic phosphate has little effect on the 2,3-DPG phosphatase activity of PGM, even at concentrations as high as 50 mM. Similarly, 25 microM Vi has little effect on the phosphatase activity. However, in the presence of 25 microM Vi, a phosphate concentration of 20 mM increases the phosphatase activity by more than 3-fold. This behavior is rationalized in terms of activation of the phosphatase activity by a phosphate/vanadate mixed anhydride. This interpretation is supported by the observation of strong activation of the phosphatase activity by inorganic pyrophosphate. A molecular mechanism for the observed effects of vanadate is proposed, and the relevance of this study to the possible use of vanadate as a therapeutic agent for the treatment of sickle cell anemia is discussed.

Interaction of vanadate with phenol and tyrosine: implications for the effects of vanadate on systems regulated by tyrosine phosphorylation.

The interaction of vanadate with phenol and N-acetyltyrosine ethyl ester in aqueous solution has been studied by using 51V nuclear magnetic resonance spectroscopy. On the basis of these studies, it has been concluded that vanadate rapidly esterifies the hydroxyl group of the aromatic ring to yield a phenyl vanadate. For phenol, the equilibrium constant for this reaction in terms of the convention that the activity of liquid water is 1.0 is K1 = [phenyl vanadate]/[phenol][vanadate] = 0.97 +/- 0.02. This value is well over 4 orders of magnitude larger than estimates from the literature for the corresponding equilibrium constant for the esterification of phenol by phosphate. The equilibrium constant for esterification of the phenol moiety of N-acetyltyrosine ethyl ester is similar to that for esterification of phenol. The relevance of these observations to processes that are regulated by reversible phosphorylation/dephosphorylation of tyrosine residues is discussed, in particular the insulin-like effect of vanadate.