A 13C-n.m.r. investigation of the ionizations within an inhibitor–α-chymotrypsin complex. Evidence that both α-chymotrypsin and trypsin stabilize a hemiketal oxyanion by similar mechanisms

Finucane, Michael D.; Hudson, E A; Malthouse, J. Paul G.

doi:10.1042/bj2580853

Cited by 31 publications

(108 citation statements)

References 41 publications

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“…Spectra were recorded at 25°C. Chemical shifts in aqueous media were referenced to tetramethylsilane at 0 ppm as described previously [32]. Data were processed with no zero filling using Bruker WINNMR software and exponential multiplications with 2 Hz line broadening were carried out.…”

Section: Methodsmentioning

confidence: 99%

13C NMR analysis reveals a link between L-glutamine metabolism, D-glucose metabolism and ?-glutamyl cycle activity in a clonal pancreatic beta-cell line

et al. 2003

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Aims/hypothesis. Pancreatic islet cells and clonal betacell lines can metabolise L-glutamine at high rates. The pathway of L-glutamine metabolism has traditionally been described as L-glutamine→L-glutamate→2-oxoglutarate→oxidation in TCA cycle following conversion to pyruvate. Controversially, the metabolism of D-glucose to L-glutamate in beta cells is not widely accepted. However, L-glutamate has been proposed to be a stimulation-secretion coupling factor in glucose-induced insulin secretion. We aimed to investigate the metabolism of glutamine and glucose by using 13 C NMR analysis. Methods. BRIN-BD11 cells were incubated in the presence of 16.7 mmol/l [1-13 C]glucose, 2 mmol/l [2-13 C]L-glycine or 2 mmol/l [1,2-13 C]glutamine in the presence or absence of other amino acids or inhibitors. After an incubation period the cellular metabolites were extracted using a PCA extract procedure. After neutralisation, the extracts were prepared for analysis using 13 C-NMR spectroscopy.Results. Using 13 C NMR analysis we have shown that L-glutamine could be metabolised in BRIN-BD11 cells via reactions constituting part of the γ-glutamyl cycle producing glutathione. Moderate or high activities of the enzymes required for these pathways of metabolism including glutaminase, γ-glutamyltransferase and γ-glutamylcysteine synthetase were observed. We additionally report significant D-glucose metabolism to L-glutamate. Addition of the aminotransferase inhibitor, aminooxyacetate, attenuated Lglutamate production from D-glucose. Conclusion/interpretation. We propose that L-glutamine metabolism is important in the beta cell for generation of stimulus-secretion coupling factors, precursors of glutathione synthesis and for supplying carbon for oxidation in the TCA cycle. D-glucose, under appropriate conditions, can be converted to L-glutamate via an aminotransferase catalysed step. [Diabetologia (2003[Diabetologia ( ) 46:1512[Diabetologia ( -1521 Keywords Pancreatic beta cells, NMR, γ-glutamyl cycle, L-glutamine, L-glutamate, glutathione. Diabetologia (2003( ) 46:1512( -1521( DOI 10.1007( /s00125-003-1184 13 C NMR analysis reveals a link between L-glutamine metabolism, D-glucose metabolism and γ-glutamyl cycle activity in a clonal pancreatic beta-cell line

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Section: Methodsmentioning

confidence: 99%

13C NMR analysis reveals a link between L-glutamine metabolism, D-glucose metabolism and ?-glutamyl cycle activity in a clonal pancreatic beta-cell line

et al. 2003

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“…In aqueous solutions the chemical shift of the alpha-carbon of glycine was used as a chemical reference as described previously [14]. The chemical shift d 6 -dimethyl sulphoxide (< 5%…”

Section: Nmr Spectroscopymentioning

confidence: 99%

“…Using 13 C-NMR it has been shown that when substrate derived chloromethylketone inhibitors alkylate the active site histidine residue of the serine proteases trypsin [12,13], chymotrypsin [10,11,[14][15][16] and subtilisin [10,11,16] the active site serine hydroxy group reacts with ketone carbon to form a tetrahedral adduct analogous to the catalytic tetrahedral intermediate.…”

Section: Introductionmentioning

confidence: 99%

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

Howe

Rogers

Hewage

et al. 2009

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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“…Inhibitor Synthesis-Z-Ala-Pro-Phe-glyoxal, Z-Ala-ProPhe- [2][3][4][5][6][7][8][9][10][11][12][13] C]glyoxal, Z-Ala-Ala-Phe-glyoxal, Z-Ala-Ala-[1-13 C]Phe-glyoxal, and Z-Ala-Ala-Phe- [2-13 C]glyoxal were synthesized using the methods described by Djurdjevic-Pahl et al (1).…”

Section: Materials-l-[1-mentioning

confidence: 99%

13C and 1H NMR Studies of Ionizations and Hydrogen Bonding in Chymotrypsin-Glyoxal Inhibitor Complexes

Spink

Cosgrove

Rogers

et al. 2007

Journal of Biological Chemistry

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Benzyloxycarbonyl (Z)-Ala-Pro-Phe-glyoxal and Z-Ala-AlaPhe-glyoxal have both been shown to be inhibitors of ␣-chymotrypsin with minimal K i values of 19 and 344 nM, respectively, at neutral pH. These K i values increased at low and high pH with pK a values of ϳ4.0 and ϳ10.5, 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.13 C NMR has been used to show that at low pH the glyoxal keto carbon is sp 3 -hybridized with a chemical shift of ϳ100.7 ppm and that the aldehyde carbon is hydrated with a chemical shift of ϳ91.6 ppm. The signal at ϳ100.7 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 pK a of ϳ4.5, the aldehyde carbon dehydrates to give a signal at ϳ205.5 ppm and the hemiketal forms an oxyanion at ϳ107.0 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 (pK a ϳ9.2). On binding either Z-Ala-Pro-Phe-glyoxal or Z-AlaAla-Phe-glyoxal to ␣-chymotrypsin at 4 and 25°C, 1 H NMR is used to show that the binding of these glyoxal inhibitors raises the pK a 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 pK a 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 pK a value of the oxyanion forming a zwitterionic tetrahedral intermediate during catalysis.Specific substrate-derived glyoxal inhibitors have been shown to be potent inhibitors of the serine proteinases (1-4). Z 4 -Ala-Pro-Phe-glyoxal is an extremely potent reversible inhibitor of ␦-chymotrypsin with an apparent disassociation constant of 25 Ϯ 8 nM at pH 7.0 (1).The ␣-keto carbon of the glyoxal inhibitor is expected to occupy the same position as the carbonyl carbon of a substrate, and it has been shown that it is bound as a tetrahedral adduct, which should closely resemble the tetrahedral intermediate formed during substrate catalysis (1). By using 13 C NMR, it has been shown that ␦-chymotrypsin (1) and subtilisin (2) reduce the oxyanion pK a by ϳ6 and ϳ8 pK a units, respectively. It has been estimated that hydrogen bonding in the oxyanion hole will only reduce the oxyanion pK a by ϳ1.3 pK a units (1). This is consistent with the fact that hydrogen bonding is expected to be effective in both water and in the oxyanion hole, and so it should not reduce the oxyanion pK a to a value lower than that expected in water. This has led to the conclusion that hydrogen bonding in the oxyanion hole only has a minor role in lowering the oxyanion pK a (5-7). However, it has been proposed that substrate binding raises the pK a of the imidazolium ion of the active site histidine enabling ...

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A 13C-n.m.r. investigation of the ionizations within an inhibitor–α-chymotrypsin complex. Evidence that both α-chymotrypsin and trypsin stabilize a hemiketal oxyanion by similar mechanisms

Cited by 31 publications

References 41 publications

13C NMR analysis reveals a link between L-glutamine metabolism, D-glucose metabolism and ?-glutamyl cycle activity in a clonal pancreatic beta-cell line

13C NMR analysis reveals a link between L-glutamine metabolism, D-glucose metabolism and ?-glutamyl cycle activity in a clonal pancreatic beta-cell line

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

13C and 1H NMR Studies of Ionizations and Hydrogen Bonding in Chymotrypsin-Glyoxal Inhibitor Complexes

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