Correction to Kinetic Study of the Effect of pH on the Trypsin-Catalyzed Hydrolysis of N-α-benzyloxycarbonyl-l-lysine-p-nitroanilide: Mechanism of Trypsin Catalysis.

[This corrects the article DOI: 10.1021/acsomega.9b03750.].

Kinetic Studies of the Effect of pH on the Trypsin-Catalyzed Hydrolysis of N-α-benzyloxycarbonyl-l-lysine-p-nitroanilide: Mechanism of Trypsin Catalysis.

The pH dependence of the trypsin-catalyzed hydrolysis of N-α-benzyloxycarbonyl-l-lysine p-nitroanilide has been studied at 25 °C. k cat/K M was maximal at alkaline pH values but decreased with decreasing pH. k cat/K M was dependent on free enzyme pK a values of 6.75 ± 0.09 and 4.10 ± 0.13, which were assigned to the ionization of the active site histidine-57 and aspartate-189, respectively. Protonation of either group abolished catalytic activity. k cat is shown to equal the acylation rate constant k 2 over the pH range studied. k 2 decreased on the protonation of two groups with pK a values of 4.81 ± 0.15 and 4.23 ± 0.19. We assign the pK a of 4.23 to the ionization of the aspartate-189 residue and the pK a of 4.81 to the oxyanion of the tetrahedral intermediate formed during acylation. We conclude that during acylation, breakdown of the catalytic tetrahedral intermediate is rate-limiting and that there is a strong interaction between the imidazolium ion of histidine-57 and the oxyanion of the catalytic tetrahedral intermediate, which perturbs their pK a values. From the pH dependence of k 3, we conclude that deacylation depends on a pK a of 6.41 ± 0.22 and that the ionization of the carboxylate group of aspartate-189 does not have a significant effect on the rate of deacylation (k 3). A catalytic mechanism is proposed to explain the pH dependence of catalysis.

A new lysine derived glyoxal inhibitor of trypsin, its properties and utilization for studying the stabilization of tetrahedral adducts by trypsin.

New trypsin inhibitors Z-Lys-COCHO and Z-Lys-H have been synthesised. Ki values for Z-Lys-COCHO, Z-Lys-COOH, Z-Lys-H and Z-Arg-COOH have been determined. The glyoxal group (-COCHO) of Z-Lys-COCHO increases binding ~300 fold compared to Z-Lys-H. The α-carboxylate of Z-Lys-COOH has no significant effect on inhibitor binding. Z-Arg-COOH is shown to bind ~2 times more tightly than Z-Lys-COOH. Both Z-Lys-13COCHO and Z-Lys-CO13CHO have been synthesized. Using Z-Lys-13COCHO we have observed a signal at 107.4 ppm by 13C NMR which is assigned to a terahedral adduct formed between the hydroxyl group of the catalytic serine residue and the 13C-enriched keto-carbon of the inhibitor glyoxal group. Z-Lys-CO13CHO has been used to show that in this tetrahedral adduct the glyoxal aldehyde carbon is not hydrated and has a chemical shift of 205.3 ppm. Hemiketal stabilization is similar for trypsin, chymotrypsin and subtilisin Carlsberg. For trypsin hemiketal formation is optimal at pH 7.2 but decreases at pHs 5.0 and 10.3. The effective molarity of the active site serine hydroxyl group of trypsin is shown to be 25300 M. At pH 10.3 the free glyoxal inhibitor rapidly (t1/2=0.15 h) forms a Schiff base while at pH 7 Schiff base formation is much slower (t1/2=23 h). Subsequently a free enol species is formed which breaks down to form an alcohol product. These reactions are prevented in the presence of trypsin and when the inhibitor is bound to trypsin it undergoes an internal Cannizzaro reaction via a C2 to C1 alkyl shift producing an α-hydroxycarboxylic acid.

Quantifying tetrahedral adduct formation and stabilization in the cysteine and the serine proteases.

Two new papain inhibitors have been synthesized where the terminal α-carboxyl groups of Z-Phe-Ala-COOH and Ac-Phe-Gly-COOH have been replaced by a proton to give Z-Phe-Ala-H and Ac-Phe-Gly-H. We show that for papain, replacing the terminal carboxylate group of a peptide inhibitor with a hydrogen atom decreases binding 3-4 fold while replacing an aldehyde or glyoxal group with a hydrogen atom decreases binding by 300,000-1,000,000 fold. Thiohemiacetal formation by papain with aldehyde or glyoxal inhibitors is shown to be ~10,000 times more effective than hemiacetal or hemiketal formation with chymotrypsin. It is shown using effective molarities, that for papain, thiohemiacetal stabilization is more effective with aldehyde inhibitors than with glyoxal inhibitors. The effective molarity obtained when papain is inhibited by an aldehyde inhibitor is similar to the effective molarity obtained when chymotrypsin is inhibited by glyoxal inhibitors showing that both enzymes can stabilize tetrahedral adducts by similar amounts. Therefore the greater potency of aldehyde and glyoxal inhibitors with papain is not due to greater thiohemiacetal stabilization by papain compared to the hemiketal and hemiacetal stabilization by chymotrypsin, instead it reflects the greater intrinsic reactivity of the catalytic thiol group of papain compared to the catalytic hydroxyl group of chymotrypsin. It is argued that while the hemiacetals and thiohemiacetals formed with the serine and cysteine proteases respectively can mimic the catalytic tetrahedral intermediate they are also analogues of the productive and non-productive acyl intermediates that can be formed with the cysteine and serine proteases.

A 13C-NMR study of azacryptand complexes.

An azacryptand has been solubilised in aqueous media containing 50% (v/v) dimethyl sulphoxide. (13)C-NMR has been used to determine how the azacryptand is affected by zinc binding at pH 10. Using (13)C-NMR and (13)C-enriched bicarbonate we have been able to observe the formation of 4 different carbamate derivatives of the azacryptand at pH 10. The azacryptand was shown to solubilise zinc or cadmium at alkaline pHs. Two moles of zinc are bound per mole of azacryptand and this complex binds 1 mole of carbonate. By replacing the zinc with cadmium-113 we have shown that the (13)C-NMR signal of the (13)C-enriched carbon of the bound carbonate is split into two triplets at 2.2 °C. This shows that two cadmium complexes are formed and in each of these complexes the carbonate group is bound by two magnetically equivalent metal ions. It also demonstrates that these cadmium complexes are not in fast exchange. From temperature studies we show that in the zinc complexes both complexes are in fast exchange with each other but are in slow exchange with free bicarbonate. HOESY is used to determine the position of the carbonate carbon in the complex. The solution and crystal structures of the zinc-carbonate-azacryptand complexes are compared.

Hemiacetal stabilization in a chymotrypsin inhibitor complex and the reactivity of the hydroxyl group of the catalytic serine residue of chymotrypsin.

The aldehyde inhibitor Z-Ala-Ala-Phe-CHO has been synthesized and shown by (13)C-NMR to react with the active site serine hydroxyl group of alpha-chymotrypsin to form two diastereomeric hemiacetals. For both hemiacetals oxyanion formation occurs with a pKa value of ~7 showing that chymotrypsin reduces the oxyanion pKa values by ~5.6 pKa units and stabilizes the oxyanions of both diastereoisomers by ~32kJmol(-1). As pH has only a small effect on binding we conclude that oxyanion formation does not have a significant effect on binding the aldehyde inhibitor. By comparing the binding of Z-Ala-Ala-Phe-CHO with that of Z-Ala-Ala-Phe-H we estimate that the aldehyde group increases binding ~100 fold. At pH7.2 the effective molarity of the active site serine hydroxy group is ~6000 which is ~7× less effective than with the corresponding glyoxal inhibitor. Using (1)H-NMR we have shown that at both 4 and 25°C the histidine pKa is ~7.3 in free chymotrypsin and it is raised to ~8 when Z-Ala-Ala-Phe-CHO is bound. We conclude that oxyanion formation only has a minor role in raising the histidine pKa and that the aldehyde hydrogen must be replaced by a larger group to raise the histidine pKa>10 and give stereospecific formation of tetrahedral intermediates. The results show that a large increase in the pKa of the active site histidine is not needed for the active site serine hydroxyl group to have an effective molarity of 6000.

Vinyl sulfone-based peptidomimetics as anti-trypanosomal agents: design, synthesis, biological and computational evaluation.

A series of vinyl sulfone-containing peptidomimetics were rationally designed, synthesized, and evaluated against Trypanosoma brucei brucei . These electrophilic compounds are likely to exert their antitrypanosomal activity via inhibition of trypanosomal cysteine proteases, TbCatB and rhodesain, through alkylation of a key cysteine residue within the protease active site. The series was designed to present complementary groups to naturally recognized peptide substrates while probing tolerance to a range of substitutions at the P1, P1', and P2 positions. The most potent compound, 29 (EC50 = 70 nM, T. b. brucei whole cell assay), displayed minimal toxicity (>785 times selectivity) when assayed for cytotoxicity against the human promyelocytic leukemia (HL-60) cell line. Cells treated with compound 29, as with K777 (2), exhibited an increase in both the number of multinucleated cells and cells with swollen flagellar pockets. Computational analysis revealed a strong correlation between the hypothetical binding mode in TbCatB/rhodesain and trypanocidal activity in vitro.

Importance of tetrahedral intermediate formation in the catalytic mechanism of the serine proteases chymotrypsin and subtilisin.

Two new inhibitors in which the terminal α-carboxyl groups of Z-Ala-Ala-Phe-COOH and Z-Ala-Pro-Phe-COOH have been replaced with a proton to give Z-Ala-Ala-Phe-H and Z-Ala-Pro-Phe-H, respectively, have been synthesized. Using these inhibitors, we estimate that for α-chymotrypsin and subtilisin Carlsberg the terminal carboxylate group decreases the level of inhibitor binding 3-4-fold while a glyoxal group increases the level of binding by 500-2000-fold. We show that at pH 7.2 the effective molarities of the catalytic hydroxyl group of the active site serine are 41000-229000 and 101000-159000 for α-chymotrypsin and subtilisin Carlsberg, respectively. It is estimated that oxyanion stabilization and the increased effective molarity of the catalytic serine hydroxyl group can account for the catalytic efficiency of the reaction. We argue that substrate binding induces the formation of a strong hydrogen bond or low-barrier hydrogen bond between histidine-57 and aspartate-102 that increases the pK(a) of the active site histidine, allowing it to be an effective general base catalyst for the formation of the tetrahedral intermediate and increasing the effective molarity of the catalytic hydroxyl group of serine-195. A catalytic mechanism for acyl intermediate formation in the serine proteases is proposed.

Mechanism of the binding of Z-L-tryptophan and Z-L-phenylalanine to thermolysin and stromelysin-1 in aqueous solutions.

The chemical shift of the carboxylate carbon of Z-tryptophan is increased from 179.85 to 182.82 ppm and 182.87 ppm on binding to thermolysin and stromelysin-1 respectively. The chemical shift of Z-phenylalanine is also increased from 179.5 ppm to 182.9 ppm on binding to thermolysin. From pH studies we conclude that the pK(a) of the inhibitor carboxylate group is lowered by at least 1.5 pK(a) units when it binds to either enzyme. The signal at ~183 ppm is no longer observed when the active site zinc atom of thermolysin or stromelysin-1 is replaced by cobalt. We estimate that the distance of the carboxylate carbon of Z-[1-(13)C]-L-tryptophan is ≤3.71Å from the active site cobalt atom of thermolysin. We conclude that the side chain of Z-[1-(13)C]-L-tryptophan is not bound in the S(2)' subsite of thermolysin. As the chemical shifts of the carboxylate carbons of the bound inhibitors are all ~183 ppm we conclude that they are all bound in a similar way most probably with the inhibitor carboxylate group directly coordinated to the active site zinc atom. Our spectrophotometric results confirm that the active site zinc atom is tetrahedrally coordinated when the inhibitors Z-tryptophan or Z-phenylalanine are bound to thermolysin.

pH stability of the stromelysin-1 catalytic domain and its mechanism of interaction with a glyoxal inhibitor.

The stromelysin-1 catalytic domain(83-247) (SCD) is stable for at least 16 h at pHs 6.0-8.4. At pHs 5.0 and 9.0 there is exponential irreversible denaturation with half lives of 38 and 68 min respectively. At pHs 4.5 and 10.0 irreversible denaturation is biphasic. At 25°C, C-terminal truncation of stromelysin-1 decreases the stability of the stromelysin-1 catalytic domain at pH values >8.4 and <6.0. We describe the conversion of the carboxylate group of (ßR)-ß-[[[(1S)-1-[[[(1S)-2-Methoxy-1-phenylethyl]amino]carbonyl]-2,2-dimethylpropyl]amino]carbonyl]-2-methyl-[1,1'-biphenyl]-4-hexanoic acid (UK-370106-COOH) a potent inhibitor of the metalloprotease stromelysin-1 to a glyoxal group (UK-370106-CO(13)CHO). At pH 5.5-6.5 the glyoxal inhibitor is a potent inhibitor of stromelysin-1 (K(i)=~1µM). The aldehyde carbon of the glyoxal inhibitor was enriched with carbon-13 and using carbon-13 NMR we show that the glyoxal aldehyde carbon is fully hydrated when it is in aqueous solutions (90.4ppm) and also when it is bound to SCD (~92.0ppm). We conclude that the hemiacetal hydroxyl groups of the glyoxal inhibitor are not ionised when the glyoxal inhibitor is bound to SCD. The free enzyme pK(a) values associated with inhibitor binding were 5.9 and 6.2. The formation and breakdown of the signal at ~92ppm due to the bound UK-370106-CO(13)CHO inhibitor depends on pK(a) values of 5.8 and 7.8 respectively. No strong hydrogen bonds are present in free SCD or in SCD-inhibitor complexes. We conclude that the inhibitor glyoxal group is not directly coordinated to the catalytic zinc atom of SCD.

Conformational, receptor interaction and alanine scan studies of glucose-dependent insulinotropic polypeptide.

Glucose-dependent insulinotropic polypeptide (GIP) is an insulinotropic incretin hormone that stimulates insulin secretion during a meal. GIP has glucose lowering abilities and hence is considered as a potential target molecule for type 2 diabetes therapy. In this article, we present the solution structure of GIP in membrane-mimicking environments by proton NMR spectroscopy and molecular modelling. GIP adopts an α-helical conformation between residues Phe(6)-Gly(31) and Ala(13)-Gln(29) for micellar and bicellar media, respectively. Previously we examined the effect of N-terminal Ala substitution in GIP, but here eight GIP analogues were synthesised by replacing individual residues within the central 8-18 region with alanine. These studies showed relatively minor changes in biological activity as assessed by insulin releasing potency. However, at higher concentration, GIP(Ala(16)), and GIP(Ala(18)) showed insulin secreting activity higher than the native GIP (P<0.01 to P<0.001) in cultured pancreatic BRIN-BD11 cells. Receptor interaction studies of the native GIP with the extracellular domain of its receptor were performed by using two different docking algorithms. At the optimised docking conformation, the complex was stabilised by the presence of hydrophobic interactions and intermolecular hydrogen bonding. Further, we have identified some potentially important additional C-terminal interactions of GIP with its N-terminal extracellular receptor domain.

Oxyanion and tetrahedral intermediate stabilisation by subtilisin: detection of a new tetrahedral adduct.

The peptide-derived glyoxal inhibitor Z-Ala-Ala-Phe-glyoxal has been shown to be approximately 10 fold more effective as an inhibitor of subtilisin than Z-Ala-Pro-Phe-glyoxal. Signals at 107.2 ppm and 200.5 ppm are observed for the glyoxal keto and aldehyde carbons of the inhibitor bound to subtilisin, showing that the glyoxal keto and aldehyde carbons are sp(3) and sp(2) hybridised respectively. The signal at 107.2 ppm from the carbon atom attached to the hemiketal oxyanion is formed in a slow exchange process that involves the dehydration of the glyoxal aldehyde carbon. Two additional signals are observed one at 108.2 ppm and the other at 90.9 ppm for the glyoxal keto and aldehyde carbons respectively at pHs 6-8 demonstrating that subtilisin forms an additional tetrahedral adduct with Z-Ala-Ala-Phe-glyoxal in which both the glyoxal keto and aldehyde carbons are sp(3) hybridised. For the first time we can quantify oxyanion stabilisation in subtilisin. We conclude that oxyanion stabilisation is more effective in subtilisin than in chymotrypsin. Using (1)H-NMR we show that the binding of Z-Ala-Ala-Phe-glyoxal to subtilisin raises the pK(a) of the imidazolium ion of the active site histidine residue promoting oxyanion stabilisation. The mechanistic significance of these results is discussed.

Prolonged L-alanine exposure induces changes in metabolism, Ca(2+) handling and desensitization of insulin secretion in clonal pancreatic beta-cells.

Acute insulin-releasing actions of amino acids have been studied in detail, but comparatively little is known about the beta-cell effects of long-term exposure to amino acids. The present study examined the effects of prolonged exposure of beta-cells to the metabolizable amino acid L-alanine. Basal insulin release or cellular insulin content were not significantly altered by alanine culture, but acute alanine-induced insulin secretion was suppressed by 74% (P<0.001). Acute stimulation of insulin secretion with glucose, KCl or KIC (2-oxoisocaproic acid) following alanine culture was not affected. Acute alanine exposure evoked strong cellular depolarization after control culture, whereas AUC (area under the curve) analysis revealed significant (P<0.01) suppression of this action after culture with alanine. Compared with control cells, prior exposure to alanine also markedly decreased (P<0.01) the acute elevation of [Ca(2+)](i) (intracellular [Ca(2+)]) induced by acute alanine exposure. These diminished stimulatory responses were partially restored after 18 h of culture in the absence of alanine, indicating reversible amino-acid-induced desensitization. (13)C NMR spectra revealed that alanine culture increased glutamate labelling at position C4 (by 60%; P<0.01), as a result of an increase in the singlet peak, indicating increased flux through pyruvate dehydrogenase. Consistent with this, protein expression of the pyruvate dehydrogenase kinases PDK2 and PDK4 was significantly reduced. This was accompanied by a decrease in cellular ATP (P<0.05), consistent with diminished insulin-releasing actions of this amino acid. Collectively, these results illustrate the phenomenon of beta-cell desensitization by amino acids, indicating that prolonged exposure to alanine can induce reversible alterations to metabolic flux, Ca(2+) handling and insulin secretion.

Determination of the structure of tetrahedral transition state analogues bound at the active site of chymotrypsin using 18O and 2H isotope shifts in the 13C NMR spectra of glyoxal inhibitors.

The peptide-derived glyoxal inhibitor Z-Ala-Pro-Phe-glyoxal, where Z is benzyloxycarbonyl, is an extremely potent inhibitor of chymotrypsin. When it is bound to chymotrypsin both the glyoxal (RCOCHO) keto and aldehyde carbons are sp3 hybridized with chemical shifts of 100.7 and 91.4 ppm, respectively. However it is has not been shown whether these carbons are bound as hydrates or whether the active-site serine has reacted with them to form the corresponding hemiketal or hemiacetal. In this study we use 18O isotope shifts to determine whether one or two exchangeable oxygen atoms are attached to the glyoxal keto or aldehyde carbons when it is free in water or bound to alpha-chymotrypsin. Both the 18O isotope shifts at the free and enzyme-bound aldehyde carbons were approximately 0.04 ppm showing that it is hydrated in both the free and bound forms. The 18O isotope shift for the free hydrated keto carbon at 96.6 ppm was 0.046-0.049 ppm, but this was reduced to 0.026 ppm when the glyoxal inhibitor was bound to alpha-chymotrypsin showing that the nonexchangeable serine hydroxyl group has formed a hemiketal with glyoxal keto carbon. Deuterium isotope shifts on the 13C NMR signals from the glyoxal inhibitor when it free and hydrated, when it is bound to chymotrypsin, as well as when it forms a model hemiketal confirm that the serine hydroxyl group has formed a hemiketal with the glyoxal keto carbon. The reasons for the different reaction specificities of glyoxal inhibitors for the active-site nucleophiles of serine and cysteine proteases are discussed.

NMR study of the inhibition of pepsin by glyoxal inhibitors: mechanism of tetrahedral intermediate stabilization by the aspartyl proteases.

Z-Ala-Ala-Phe-glyoxal (where Z is benzyloxycarbonyl) has been shown to be a competitive inhibitor of pepsin with a Ki = 89 +/- 24 nM at pH 2.0 and 25 degrees C. Both the ketone carbon (R13COCHO) and the aldehyde carbon (RCO13CHO) of the glyoxal group of Z-Ala-Ala-Phe-glyoxal have been 13C-enriched. Using 13C NMR, it has been shown that when the inhibitor is bound to pepsin, the glyoxal keto and aldehyde carbons give signals at 98.8 and 90.9 ppm, respectively. This demonstrates that pepsin binds and preferentially stabilizes the fully hydrated form of the glyoxal inhibitor Z-Ala-Ala-Phe-glyoxal. From 13C NMR pH studies with glyoxal inhibitor, we obtain no evidence for its hemiketal or hemiacetal hydroxyl groups ionizing to give oxyanions. We conclude that if an oxyanion is formed its pKa must be >8.0. Using 1H NMR, we observe four hydrogen bonds in free pepsin and in pepsin/Z-Ala-Ala-Phe-glyoxal complexes. In the pepsin/pepstatin complex an additional hydrogen bond is formed. We examine the effect of pH on hydrogen bond formation, but we do not find any evidence for low-barrier hydrogen bond formation in the inhibitor complexes. We conclude that the primary role of hydrogen bonding to catalytic tetrahedral intermediates in the aspartyl proteases is to correctly orientate the tetrahedral intermediate for catalysis.

The bioactive conformation of glucose-dependent insulinotropic polypeptide by NMR and CD spectroscopy.

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal incretin hormone, which modulates physiological insulin secretion. Because of its glucose-sensitive insulinotropic activity, there has been a considerable interest in utilizing the hormone as a potential treatment for type 2 diabetes. Structural parameters obtained from NMR spectroscopy combined with molecular modeling techniques play a vital role in the design of new therapeutic drugs. Therefore, to understand the structural requirements for the biological activity of GIP, the solution structure of GIP was investigated by circular dichroism (CD) followed by proton nuclear magnetic resonance (NMR) spectroscopy. CD studies showed an increase in the helical character of the peptide with increasing concentration of trifluoroethanol (TFE) up to 50%. Therefore, the solution structure of GIP in 50% TFE was determined. It was found that there was an alpha-helix between residues 6 and 29, which tends to extend further up to residue 36. The implications of the C-terminal extended helical segment in the inhibitory properties of GIP on gastric acid secretion are discussed. It is shown that the adoption by GIP of an alpha-helical secondary structure is a requirement for its biological activity. Knowledge of the solution structure of GIP will help in the understanding of how the peptide interacts with its receptor and aids in the design of new therapeutic agents useful for the treatment of diabetes.

13C and 1H NMR studies of ionizations and hydrogen bonding in chymotrypsin-glyoxal inhibitor complexes.

Benzyloxycarbonyl (Z)-Ala-Pro-Phe-glyoxal and Z-Ala-Ala-Phe-glyoxal have both been shown to be inhibitors of alpha-chymotrypsin with minimal Ki values of 19 and 344 nM, respectively, at neutral pH. These Ki values increased at low and high pH with pKa values of approximately 4.0 and approximately 10.5, respectively. By using surface plasmon resonance, we show that the apparent association rate constant for Z-Ala-Pro-Phe-glyoxal is much lower than the value expected for a diffusion-controlled reaction. 13C NMR has been used to show that at low pH the glyoxal keto carbon is sp3-hybridized with a chemical shift of approximately 100.7 ppm and that the aldehyde carbon is hydrated with a chemical shift of approximately 91.6 ppm. The signal at approximately 100.7 ppm is assigned to the hemiketal formed between the hydroxy group of serine 195 and the keto carbon of the glyoxal. In a slow exchange process controlled by a pKa of approximately 4.5, the aldehyde carbon dehydrates to give a signal at approximately 205.5 ppm and the hemiketal forms an oxyanion at approximately 107.0 ppm. At higher pH, the re-hydration of the glyoxal aldehyde carbon leads to the signal at 107 ppm being replaced by a signal at 104 ppm (pKa approximately 9.2). On binding either Z-Ala-Pro-Phe-glyoxal or Z-Ala-Ala-Phe-glyoxal to alpha-chymotrypsin at 4 and 25 degrees C, 1H NMR is used to show that the binding of these glyoxal inhibitors raises the pKa value of the imidazolium ion of histidine 57 to a value of >11 at both 4 and 25 degrees C. We discuss the mechanistic significance of these results, and we propose that it is ligand binding that raises the pKa value of the imidazolium ring of histidine 57 allowing it to enhance the nucleophilicity of the hydroxy group of the active site serine 195 and lower the pKa value of the oxyanion forming a zwitterionic tetrahedral intermediate during catalysis.

Effect of acute dietary standardization on the urinary, plasma, and salivary metabolomic profiles of healthy humans.

BACKGROUND: Metabolomics in human nutrition research is faced with the challenge that changes in metabolic profiles resulting from diet may be difficult to differentiate from normal physiologic variation. OBJECTIVE: We assessed the extent of intra- and interindividual variation in normal human metabolic profiles and investigated the effect of standardizing diet on reducing variation. DESIGN: Urine, plasma, and saliva were collected from 30 healthy volunteers (23 females, 7 males) on 4 separate mornings. For visits 1 and 2, free food choice was permitted on the day before biofluid collection. Food choice on the day before visit 3 was intended to mimic that for visit 2, and all foods were standardized on the day before visit 4. Samples were analyzed by using 1H nuclear magnetic resonance spectroscopy followed by multivariate data analysis. RESULTS: Intra- and interindividual variations were considerable for each biofluid. Visual inspection of the principal components analysis scores plots indicated a reduction in interindividual variation in urine, but not in plasma or saliva, after the standard diet. Partial least-squares discriminant analysis indicated time-dependent changes in urinary and salivary samples, mainly resulting from creatinine in urine and acetate in saliva. The predictive power of each model to classify the samples as either night or morning was 85% for urine and 75% for saliva. CONCLUSIONS: Urine represented a sensitive metabolic profile that reflected acute dietary intake, whereas plasma and saliva did not. Future metabolomics studies should consider recent dietary intake and time of sample collection as a means of reducing normal physiologic variation.

NMR and alanine scan studies of glucose-dependent insulinotropic polypeptide in water.

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone that stimulates the secretion of insulin after ingestion of food. GIP also promotes the synthesis of fatty acids in adipose tissue. Therefore, it is not surprising that numerous literature reports have shown that GIP is linked to diabetes and obesity-related diseases. In this study, we present the solution structure of GIP in water determined by NMR spectroscopy. The calculated structure is characterized by the presence of an alpha-helical motif between residues Ser(11) and Gln(29). The helical conformation of GIP is further supported by CD spectroscopic studies. Six GIP-(1-42)Ala(1-7) analogues were synthesized by replacing individual N-terminal residues with alanine. Alanine scan studies of these N-terminal residues showed that the GIP-(1-42)Ala(6) was the only analogue to show insulin-secreting activity similar to that of the native GIP. However, when compared with glucose, its insulinotropic ability was reduced. For the first time, these NMR and modeling results contribute to the understanding of the structural requirements for the biological activity of GIP.

Impact of the gliotoxin L-serine-O-sulphate on cellular metabolism in cultured rat astrocytes.

L-serine-O-sulphate is a member of a group of amino acids collectively called gliotoxins and is a substrate for the high affinity sodium-dependent glutamate transporters. Previous studies have shown that it is toxic to primary cultures of astrocytes but the mode of toxicity is unknown. The current study demonstrates that L-serine-O-sulphate, at a sub-toxic concentration (400 microM), causes significant disruption to glucose and alanine metabolism in cultures of rat cortical astrocytes. More specifically, using (13)C NMR spectroscopy a significant reduction in labelled end products from [1-(13)C]glucose and [3-(13)C]alanine was found in the presence of L-serine-O-sulphate. Additionally, using [2-(13)C]glycine a 27% reduction in de novo glutathione synthesis was observed in the presence of the gliotoxin. Incubation of the cells with L-serine-O-sulphate reduced the activity of alanine and aspartate aminotransferase by 53% and 67%, respectively. Collectively these results show that the gliotoxin, L-serine-O-sulphate, causes major disruptions to metabolic pathways in primary cultures of astrocytes.