J. Paul G. Malthouse

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 α‐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 α‐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. Proteins 2007.

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 α-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 ∼4.0 and ∼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 ∼100.7 ppm and that the aldehyde carbon is hydrated with a chemical shift of ∼91.6 ppm. The signal at ∼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 ∼4.5, the aldehyde carbon dehydrates to give a signal at ∼205.5 ppm and the hemiketal forms an oxyanion at ∼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 ∼9.2). On binding either Z-Ala-Pro-Phe-glyoxal or Z-Ala-Ala-Phe-glyoxal to α-chymotrypsin at 4 and 25 °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 °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.

Prolonged L-alanine exposure induces changes in metabolism, Ca2+ 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.

13C-NMR study of the inhibition of delta-chymotrypsin by a tripeptide-glyoxal inhibitor.

A new inhibitor, Z-Ala-Pro-Phe-glyoxal (where Z is benzyloxycarbonyl),has been synthesized and shown to be a competitive inhibitor of delta-chymotrypsin, with a K(i) of 25+/-8 nM at pH 7.0 and 25 degrees C. Z-Ala-Pro-[1-(13)C]Phe-glyoxal and Z-Ala-Pro-[2-(13)C]Phe-glyoxal have been synthesized, and (13)C-NMR has been used to determine how they interact with delta-chymotrypsin. Using Z-Ala-Pro-[2-(13)C]Phe-glyoxal we have detected a signal at 100.7 p.p.m. which we assign to the tetrahedral adduct formed between the hydroxy group of Ser-195 and the (13)C-enriched keto-carbon of the inhibitor. This signal is in a pH-dependent slow exchange with a signal at 107.6 p.p.m. which depends on a pK(a) of approximately 4.5, which we assign to oxyanion formation. Thus we are the first to detect an oxyanion pK(a) in a reversible chymotrypsin-inhibitor complex. A smaller titration shift of 100.7 p.p.m. to 103.9 p.p.m. with a pK(a) of approximately 5.3 is also detected due to a rapid exchange process. This pK(a) is also detected with the Z-Ala-Pro-[1-(13)C]Phe-glyoxal inhibitor and gives a larger titration shift of 91.4 p.p.m. to 97.3 p.p.m., which we assign to the ionization of the hydrated aldehyde hydroxy groups of the enzyme-bound inhibitor. Protonation of the oxyanion in the oxyanion hole decreases the binding efficiency of the inhibitor. From this decrease in binding efficiency we estimate that oxyanion binding in the oxyanion hole reduces the oxyanion pK(a) by 1.3 pK(a) units. We calculate that the pK(a)s of the oxyanions of the hemiketal and hydrated aldehyde moieties of the glyoxal inhibitor are both lowered by 6.4-6.9 pK(a) units on binding to chymotrypsin. Therefore we conclude that oxyanion binding in the oxyanion hole has only a minor role in decreasing the oxyanion pK(a). We also investigate how the inhibitor breaks down at alkaline pH, and how it breaks down at neutral pH in the presence of chymotrypsin.

Gliotoxins disrupt alanine metabolism and glutathione production in C6 glioma cells: a 13C NMR spectroscopic study

Gliotoxins are a group of amino acids that are toxic to astrocytes, and are substrates of high-affinity sodium-dependent glutamate transporters. In the present study, C6 glioma cells were preincubated for 20 h in the presence of 400 microM L-alpha-aminoadipate, L-serine-O-sulphate, D-aspartate or L-cysteate, as well as in the presence of the poorly transported L-glutamate uptake inhibitor, L-anti-endo-methanopyrrolidine dicarboxylate. In experiments following [3-13C]alanine metabolism, all toxins caused a decreased incorporation of label into glutamate. Production of labelled lactate changed only when cells were incubated in the presence of L-alpha-aminoadipate or L-serine-O-sulphate. Incubation with L-anti-endo-methanopyrrolidine dicarboxylate caused no change in the amount of label incorporated into either glutamate or lactate. When glutathione production was followed using 1 mM [2-13C]glycine, differential effects of the gliotoxins were revealed. Most notably, both L-serine-O-sulphate and L-alpha-aminoadipate caused significant increases in labelling of glutathione. Once again, L-anti-endo-methanopyrrolidine dicarboxylate was without effect. Overall, we have shown that the gliotoxins cause disruption to alanine metabolism and glutathione production in C6 glioma cells, but that there are notable differences in their mechanisms of action. In the absence of any disruption to metabolism by L-anti-endo-methanopyrrolidine dicarboxylate, it is concluded that their mode of action involves more than inhibition of glutamate transport.

The pyridoxal‐5′‐phosphate‐dependent catalytic antibody 15A9: its efficiency and stereospecificity in catalysing the exchange of the α‐protons of glycine

13C‐NMR has been used to follow the exchange of the α‐protons of [2‐13C]glycine in the presence of pyridoxal‐5′‐phosphate and the catalytic antibody 15A9. In the presence of antibody 15A9 the 1st order exchange rates for the rapidly exchanged proton of [2‐13C]glycine were only 25 and 150 times slower than those observed with tryptophan synthase (EC 4.2.1.20) and serine hydroxymethyltransferase (EC 2.1.2.1). The catalytic antibody increases the 1st order exchange rates of the α‐protons of [2‐13C]glycine by at least three orders of magnitude. We propose that this increase is largely due to an entropic mechanism which results from binding the glycine‐pyridoxal‐5′‐phosphate Schiff base. The 1st and 2nd order exchange rates of the pro‐2S proton have been determined but we were only able to determine the 2nd order exchange rate for the pro‐2R proton of glycine. In the presence of 50 mM glycine the antibody preferentially catalyses the exchange of the pro‐2S proton of glycine. The stereospecificity of the 2nd order exchange reaction was quantified and we discuss mechanisms which could account for the observed stereospecificity.

An NMR study of alterations in [1-13C]glucose metabolism in C6 glioma cells by gliotoxic amino acids.

A series of glutamate analogues, known as gliotoxins, are toxic to astrocytes in culture, and are inhibitors or substrates of high affinity sodium-dependent glutamate transporters. The mechanisms by which these gliotoxins cause toxicity are not fully understood. The effects of a series of gliotoxic amino acids (L-alpha-aminoadipate, L-serine-O-sulphate, D-aspartate and L-cysteate) on metabolism of [1-13C]glucose were examined in C6 glioma cells using 13C nuclear magnetic resonance (NMR) spectroscopy. The cells were preincubated in the presence of sub toxic concentrations of each gliotoxin (400 micromol/l) for 20 h. This was followed by incubation (4 h) with [1-13C]glucose (5.5 mmol/l) in the presence and absence of each gliotoxin. The incorporation of 13C label into the observed metabolites was analysed. Following preincubation with L-alpha-aminoadipate, D-aspartate, and L-serine-O-sulphate there was a significant decrease in the incorporation of 13C label into glutamate, alanine and lactate from [1-13C]glucose. In the presence of L-cysteate production of labelled glutamate was decreased, while there was no significant effect on the concentrations of labelled lactate and alanine. There was no change in the quantity of LDH released into the medium after incubation of the cells with any of the gliotoxins. Overall these results indicate that the presence of gliotoxins profoundly alters the flux of glucose to lactate, alanine, aspartate and glutamate.

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.

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

Abstract The aldehyde inhibitor Z-Ala-Ala-Phe-CHO has been synthesized and shown by 13C-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 1H-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.

The aspartate aminotransferase-catalysed exchange of the α-protons of aspartate and glutamate: the effects of the R386A and R292V mutations on this exchange reaction

1H-NMR was used to follow the aspartate aminotransferase-catalysed exchange of the alpha-protons of aspartate and glutamate. The effect of the concentrations of both the amino acids and the cognate keto acids on exchange rates was determined for wild-type and the R386A and R292V mutant forms of aspartate aminotransferase. The wild-type enzyme is found to be highly stereospecific for the exchange of the alpha-protons of L-aspartate and L-glutamate. The R386A mutation which removes the interaction of Arg-386 with the alpha-carboxylate group of aspartate causes an approximately 10,000-fold decrease in the first order exchange rate of the alpha-proton of L-aspartate. The R292V mutation which removes the interaction of Arg-292 with the beta-carboxylate group of L-aspartate and the gamma-carboxylate group of L-glutamate causes even larger decreases of 25,000- and 100,000-fold in the first order exchange rate of the alpha-proton of L-aspartate and L-glutamate respectively. Apparently both Arg-386 and Arg-292 must be present for optimal catalysis of the exchange of the alpha-protons of L-aspartate and L-glutamate, perhaps because the interaction of both these residues with the substrate is essential for inducing the closed conformation of the active site.