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- PublicationA 13C-NMR study of azacryptand complexesAn azacryptand has been solubilised in aqueous media containing 50% (v/v) dimethyl sulphoxide. 13C-NMR has been used to determine how the azacryptand is affected by zinc binding at pH 10. Using 13C-NMR and 13C-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 13C-NMR signal of the 13C-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.
414Scopus© Citations 4
- PublicationOxyanion and tetrahedral intermediate stabilization by subtilisin : detection of a new tetrahedral adductThe peptide-derived glyoxal inhibitor Z-Ala-Ala-Phe-glyoxal has been shown to be ~10 fold more effective as an inhibitor of subtilisin than Z-Ala-Pro-Phe-glyoxal. Signals at 107.2 p.p.m. and 200.5 p.p.m. are observed for the glyoxal keto and aldehyde carbons of the inhibitor bound to subtilisin, showing that the glyoxal keto and aldehyde carbons are sp3 and sp2 hybridized respectively. The signal at 107.2 p.p.m. 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 p.p.m. and the other at 90.9 p.p.m. 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 sp3 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 1H-NMR we show that the binding of Z-Ala-Ala-Phe-glyoxal to subtilisin raises the pKa of the imidazolium ion of the active site histidine residue promoting oxyanion stabilisation. The mechanistic significance of these results are discussed.
270Scopus© Citations 7