Acetophenone glycosides from thyme (Thymus vulgaris L.).

Four acetophenone glycosides were isolated from the butanol-soluble fraction of thyme extracts. Their structures were determined by spectral methods (MS, NMR, and 2D-NMR). Among them, two new compounds, 4-hydroxyacetophenone 4-O-[5-O-(3, 5-dimethoxy-4-hydroxybenzoyl)-beta-D-apiofuranosyl]-(1-->2)-beta-D -gl ucopyranoside (1) and 4-hydroxyacetophenone 4-O-[5-O-(4-hydroxybenzoyl)-beta-D-apiofuranosyl]-(1-->2)-beta-D-+ ++gluc opyranoside (2), were determined. Compound 1 showed weak cytotoxicity, inhibiting DNA synthesis of human leukemia cells.

Is a hydrophobic amino acid required to maintain the reactive V conformation of thiamin at the active center of thiamin diphosphate-requiring enzymes? Experimental and computational studies of isoleucine 415 of yeast pyruvate decarboxylase.

The residue I415 in pyruvate decarboxylase from Saccharomyces cerevisiae was substituted with a variety of uncharged side chains of varying steric requirements to test the hypothesis that this residue is responsible for supporting the V coenzyme conformation reported for this enzyme [Arjunan et al. (1996) J. Mol. Biol. 256, 590-600]. Changing the isoleucine to valine and threonine decreased the kcat value and shifted the kcat-pH profile to more alkaline values progressively, indicating that the residue at position 415 not only is important for providing the optimal transition state stabilization but also ensures correct alignment of the ionizable groups participating in catalysis. Substitutions to methionine (the residue used in pyruvate oxidase for this purpose) or leucine (the corresponding residue in transketolase) led to greatly diminished kcat values, showing that for each thiamin diphosphate-dependent enzyme an optimal hydrophobic side chain evolved to occupy this key position. Computational studies were carried out on the wild-type enzyme and the I415V, I415G, and I415A variants in both the absence and the presence of pyruvate covalently bound to C2 of the thiazolium ring (the latter is a model for the decarboxylation transition state) to determine whether the size of the side chain is critically required to maintain the V conformation. Briefly, there are sufficient conformational constraints from the binding of the diphosphate side chain and three conserved hydrogen bonds to the 4'-aminopyrimidine ring to enforce the V conformation, even in the absence of a large side chain at position 415. There appears to be increased coenzyme flexibility on substitution of Ile415 to Gly in the absence compared with the presence of bound pyruvate, suggesting that entropy contributes to the rate acceleration. The additional CH3 group in Ile compared to Val also provides increased hydrophobicity at the active center, likely contributing to the rate acceleration. The computational studies suggest that direct proton transfer to the 4'-imino nitrogen from the thiazolium C2H is eminently plausible.

Regulation of thiamin diphosphate-dependent 2-oxo acid decarboxylases by substrate and thiamin diphosphate.Mg(II) - evidence for tertiary and quaternary interactions.

The regulatory mechanism of substrate activation in yeast pyruvate decarboxylase is triggered by the interaction of pyruvic acid with C221 located on the beta domain at >20 A from the thiamin diphosphate (ThDP). To trace the putative information transfer pathway, substitutions were made at H92 on the alpha domain, across the domain divide from C221, at E91, next to H92 and hydrogen bonded to W412, the latter being intimately involved in the coenzyme binding locus. Additional substitutions were made at D28, E51, H114, H115, I415 and E477, all near the active center. The pH-dependent steady-state kinetic parameters, including the Hill coefficient, provide useful insight to this effort. In addition to C221, the residues H92, E91, E51 and H114 and H115 together appear to have a critical impact on the Hill coefficient, providing a pathway for information transfer. To study the activation by ThDP.Mg(II), variants at G231 (of the conserved GDG triplet) and at N258 and C259 (all three being part of the putative ThDP fold) of the E1 component of the Escherichia coli pyruvate dehydrogenase multienzyme complex were studied. Kinetic and spectroscopic evidence suggests that the Mg(II) ligands are very important to activation of the enzymes by cofactors.

Low barrier hydrogen bond is absent in the catalytic triads in the ground state but Is present in a transition-state complex in the prolyl oligopeptidase family of serine proteases.

High frequency proton NMR spectra for two members of the prolyl oligopeptidase class of serine proteases, prolyl oligopeptidase and oligopeptidase B, showed that resonances corresponding to the active center histidine Ndelta1H and Nepsilon2H generally observed in this region, are absent in these enzymes. However, for both enzymes, as well as with the H652A and H652Q active center variants of oligopeptidase B, there are two resonances observed in this region that could be assigned to two protonated histidines with a noncatalytic function. The results indicate that these two histidines participate in strong hydrogen bonds. The absence of resonances pertinent to the active center histidine resonances suggests the absence of a low barrier hydrogen bond between the Asp and His in these two enzymes in their ground states. Addition of the peptide boronic acid t-butoxycarbonyl-(D)Val-Leu-(L)boroArg to oligopeptidase B resulted in potent, slow binding inhibition of the enzyme and the appearance of a new resonance at 15.8 ppm, whose chemical shift is appropriate for a tetrahedral boronate complex and a low barrier hydrogen bond. The results demonstrate important dissimilarities between the active centers of the prolyl oligopeptidase class of serine proteases and the pancreatic and subtilisin classes both in the ground state and in the transition-state analog complexes.

Benzyloxycarbonylprolylprolinal, a transition-state analogue for prolyl oligopeptidase, forms a tetrahedral adduct with catalytic serine, not a reactive cysteine.

N-Benzyloxycarbonyl-l-prolyl-l-[1-13C]prolinal was synthesized starting with reduction of l-[1-13C]Pro to l-[1-13C]prolinol, followed by coupling with N-benzyloxycarbonyl-l-Pro to N-benzyloxycarbonyl-l-Pro-l-[1-13C]prolinol (Z-Pro-[1-13C]prolinol), and finally oxidation of the alcohol to the aldehyde with dimethyl sulphoxide. While the 13C NMR chemical shift of the aldehyde carbon is 202 p.p.m., that of the aldehyde hydrate is between 91.6 and 91.8 p.p.m., that of the dithiothreitol adduct is between 74.8 and 75.0 p. p.m., and that in the presence of the serine protease prolyl oligopeptidase is at 92.3 p.p.m.. The linewidth of the latter is 114 Hz, roughly consistent with the molecular mass of 80 kDa reported for the enzyme. Inverse detection experiments gave a 1H resonance at 5.29 p.p.m. with a linewidth of 80 Hz, also consistent with the expected chemical shift and linewidth for a hemiacetal bound to such a large enzyme, while the free hydrate gave resonances at 5.18 and 5. 25 p.p.m., with very much narrower linewidths. It is concluded that Z-Pro-prolinal, a putative transition-state analogue for prolyl oligopeptidase, forms a tetrahedral complex with the enzyme at its catalytic serine, rather than at a neighbouring cysteine that was found to be highly reactive according to chemical modification studies.

Three of four cysteines, including that responsible for substrate activation, are ionized at pH 6.0 in yeast pyruvate decarboxylase: evidence from Fourier transform infrared and isoelectric focusing studies.

Oligonucleotide-directed site-specific mutagenesis was carried out on pyruvate decarboxylase (EC 4.1.1.1) from Saccharomyces cerevisiae at three of the four cysteines (152, 221, and 222), the fourth (69) being buried according to X-ray crystallographic results [Arjunan et al. (1996) J. Mol. Biol. 256, 590-600]. All of the variants still retained significant activity, and all could be purified to homogeneity. FT-IR experiments were run on the C221S, C222S, C221S/C222S and C152A variants, as well as on the wild-type enzyme. There is a band present at 2557 cm-1 in the spectra of all variants and the wild-type enzyme, except in the spectrum of the C152A variant. This frequency is appropriate to a cysteine S-H stretching mode. It was therefore concluded that C152 is the only undissociated cysteine on the enzyme at pH 6.0, the pH optimum of this enzyme, whereas C221, C222, and C69 are all ionized. Isoelectric focusing experiments were carried out on all of these variants, as well as on the H92A variant (H92 is across the domain divide on the alpha domain, from C221 located on the beta domain). The variation in isoelectric points deduced from the data was consistent with removal of negative charges concomitant with the C221S, C222S, and C221S/C222S substitutions and removal of a positive charge with the H92A substitution when compared to that of the wild-type enzyme. The results of these two types of experiments are in good accord and suggest that the site of substrate activation at C221 [Baburina et al. (1994) Biochemistry 33, 5630-5635] is comprised of a Cys221S- +HHis92 ion pair, not unlike that found in papain and glyceraldehyde-3-phosphate dehydrogenase. This finding suggests that the regulatory site of this enzyme has been optimized for nucleophilic reactivity between the thiolate of C221 and the keto carbon of the 2-oxoacid.