Search

Chloromethane inhibitors of subtilisin and chymotrypsin, with one to four amino acid residues, have been synthesized. 13C NMR spectroscopy is used in studying the tetrahedral adducts formed by the inhibitors with chymotrypsin, subtilisin and trypsin. Results indicate that the amino acid residue occupying S1 subsite determines the oxyanion pKa for the subtilisin and chymotrypsin chloromethane derivatives. The binding in the S2 to S4 subsites has insignificant effects on the pKa values.

13C enriched cyanide was used to cyanylate both the thiol groups of Megasphaera elsdenii flavodoxin. An increase in the dissociation constant of the apoflavodoxin-flavin mononucleotide complex was noted. It was concluded that thiocyanate carbons will have a minimal chemical shift of 109 ppm in apolar solvents which do not contain hydrogen bond donors and that the chemical shift of a thiocyanate carbon can be used as a probe of its molecular environment. A discussion has arisen on the role of chemical shifts of the thiocyanate carbon of cyanylated apoflavodoxin, flavodoxin and other proteins and how it depends on the microenvironments of the thiocyanate group.

The reasons for different reaction specificities of the peptide-derived glyoxal inhibitor Z-Ala-Pro-Phe-glyoxal (Z = benzyloxycarbonyl), of chymotrypsin for the active-site nucleophiles of serine and cysteine proteases are discussed.

The reaction between the pyridoxal 5'-phosphate (PLP) analogue (10 mM) and glycine (10mM) in D20 buffered at neutral pD that does not give the expected products of a PLP-catalyzed reaction but rather gives a quantitative yield of the diasterioisomers from the formal Claisen-type addition of glycine to pyridoxal is reported.

In recent years nuclear magnetic resonance (NMR) spectroscopy has been used extensively to study the mode of action of various enzymes with both inhibitors and substrates. So far, reporter nuclei, [...]

Early experiments indicated that islet beta-cells substantially metabolized L-alanine but that insulin secretion was largely unaffected by the amino acid. It was subsequently demonstrated using more intricate studies that L-alanine is a strong stimulus to insulin secretion in the presence of glucose in normal rodent islets and beta-cell lines. Using (13)C nuclear magnetic resonance (NMR), we have demonstrated substantial oxidative metabolism of L-alanine by the clonal beta-cell line BRIN-BD11, with time-dependent increases in production of cellular glutamate and aspartate. Stimulatory effects of L-alanine on insulin secretion were attenuated by the inhibition of beta-cell oxidative phosphorylation using oligomycin. Additionally, we detected substantial production of lactate, alanine, and glutamate from glucose (16.7 mmol/l) after 60 min. On addition of 10 mmol/l L-alanine to a stimulus of 16.7 mmol/l glucose, the utilization rate of glucose increased approximately 2.4-fold. L-Alanine dramatically enhanced NMR-measurable aspects of glucose metabolism (both oxidative and nonoxidative). The enhanced rate of entry of glucose-derived pyruvate into the tricarboxylic acid (TCA) cycle in the presence of alanine may have stimulated rates of generation of key metabolites, including ATP, which affect the insulin secretory process. Thus L-alanine metabolism, in addition to the enhancing effect on glucose metabolism, contributes to the stimulatory effects of this amino acid on insulin secretion in vitro. [ABSTRACT FROM AUTHOR]

An 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. [ABSTRACT FROM AUTHOR]

A study was conducted to analyze pH-dependent spectroscopic changes related to the hydroquinone of FMN in flavodoxins. Flavodoxin from D. vulgaris was determined as the recombinant protein. Results indicated that a decrease in pH promotes a change in the electronic structure of the reduced flavin resulting from changes in the ionization state of the flavin and in the hydrogen-bonding network around the flavin.