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D H Pliura

Publications and source records attributed to D H Pliura.

8 recordsLinked to original sources
PubMed1993

Solid-state 13C NMR study of a transglutaminase-inhibitor adduct.

We have used solid-state 13C NMR to study the structure of the adduct resulting from the inactivation of the enzyme transglutaminase by 3-halo-4,5-dihydroisoxazoles. These inhibitors were conceived on the assumption that they would inhibit transglutaminase by attack of an enzyme active site cysteine thiol on the imine carbon of the dihydroisoxazole ring. The tetrahedral intermediate formed could then break down with the loss of the halide group and the subsequent formation of a stable imino thioether adduct. We have compared the 13C CPMAS spectra of the chloro-, bromo-, and (ethylthio)dihydroisozazole inhibitors, and the results indicate that the chemical shift of the C-3 carbon is sensitive to the nature of the heteroatom. Subtraction of the natural-abundance 13C solid-state NMR spectrum of the enzyme from that of the enzyme inactivated by C-3-labeled chlorodihydroisoxazole reveals a broad peak at 156 ppm. The chemical shift of this peak is very close to that observed for a model 3-ethylthio compound and suggests the formation of a stable imino thioether enzyme adduct. Similar results were obtained for lyophilized enzyme adducts and for frozen solutions of the enzyme adduct in the absence and presence of Ca2+. We have also compared these results with those obtained by solution NMR on an aqueous solution of the enzyme-inhibitor complex. The 13C-labeled C-3 resonance was not observed in this case.

Animals↗
PubMed1992

Comparative behaviour of calpain and cathepsin B toward peptidyl acyloxymethyl ketones, sulphonium methyl ketones and other potential inhibitors of cysteine proteinases.

Peptidyl acyloxymethyl ketones, previously established as potent inactivators of the lysosomal cysteine proteinase cathepsin B, were evaluated against smooth-muscle calpain, a member of the family of Ca(2+)-dependent cysteine proteinases. Only modest rates of time-dependent inhibition could be achieved, even with peptidyl affinity groups optimized for calpain and linked to a carboxylate leaving group of very low pKa [2,6-(CF3)2PhCOO-, pKa 0.58]. Selective inactivation of cathespin B versus calpain was consistently observed with this type of inhibitor. Examination of other potential inhibitors revealed a rank order of potency against calpain to be: peptidyl sulphonium methyl ketones > fluoromethyl ketones, diazomethyl ketones >> acyloxymethyl ketones, an order which differs sharply from that found for cathespin B.

Amino Acid Sequence↗
PubMed1992

Irreversible inhibition of transglutaminases by sulfonium methylketones: optimization of specificity and potency with omega-aminoacyl spacers.

Sulfonium methylketones, of structure Cbz-Phe-NH(CH2)nCOCH2S+ (CH3)2, n > 2, are specific and potent inactivators of transglutaminases. The length of the -(CH2)n-spacer moiety, n = 1-5, is a critical determinant for both the specificity and potency of the inactivator. The dipeptidyl analog Cbz-Phe-Gly-(CH2)nS+ (CH3)2, n = 1, is a more powerful inactivator of the thiol proteinase cathepsin B, k/K > 3 x 10(5) M-1 min-1, than of transglutaminases, ki(app)/Ki(app) < 1.5 x 10(4) M-1 min-1. In contrast, the gamma-aminobutyryl analog, n = 3, is a very potent transglutaminase inactivator with ki(app)/Ki(app) = 3.1 x 10(6) M-1 min-1, but does not inactivate cathepsin B. In cell studies, the gamma-aminobutyryl and epsilon-aminohexyl analogs inhibited the transglutaminase-mediated process of ionophore-induced cross-linked envelope formation by human malignant keratinocytes and the order of potency was related to that found for enzyme inhibition. The sulfonium methylketones, in equilibrium with the resonance stabilized ylides, are chemically inert towards glutathione under ambient conditions demonstrating the potential utility of this novel class of transglutaminase inhibitors for the study of enzyme inhibition in cellular environments.

Animals↗
PubMed1989

A new class of mechanism-based inhibitors of transglutaminase enzymes inhibits the formation of cross-linked envelopes by human malignant keratinocytes.

A series of tyrosinamidomethyl dihydrohaloisoxazole compounds, designed as mechanism-based inhibitors of bovine epidermal transglutaminase enzyme, was examined for effects on the formation of cross-linked envelopes by human SCC-9 malignant keratinocytes. Compounds inhibited ionophore-induced envelope formation in a manner that reflected their capacity to inhibit transglutaminase activity. Preincubation and inhibitor wash-out studies indicated that the inhibitor must be present at the time of cell activation by ionophore in order to inhibit envelope formation. The stereospecific nature of the inhibitory activity of these compounds on both transglutaminase activity and cross-linked envelope formation makes this class of compounds an important tool in the study of transglutaminase-mediated events at the cellular level.

Carcinoma, Squamous Cell↗
PubMed1980

Stereochemical course of a phosphokinase using a chiral [18O]phosphorothioate. Comparison with the transfer of a chiral [16O,17O,18O]phosphoryl group.

Synthetic adenosine 5'-O-[3-18O,3-thio]triphosphate having the R configuration at the gamma-phosphorus has been used as a substrate in the reaction catalyzed by glycerol kinase. The product sn-glycerol 3-[18O]phosphorothioate has been isolated, and the configuration at phosphorus has been determined by ring closure to the two diastereoisomeric cyclic 2,3-phosphorothioates of sn-glycerol and analysis of the 18O content of each diastereoisomer. The structural identity of these diastereoisomers has been determined by correlation with one of the corresponding diastereoisomers of the cyclic 2,3-phosphorothioate of D-glycerate, whose crystal structure is reported here. From these experiments it is evident that glyc:rol kinase catalyzes the transfer of a thiophosphoryl group with inversion of the configuration at phosphorus, in gratifying agreement with the result from the transfer of a chiral [16O,17O,18O]phosphoryl group [Blättler, W. A., & Knowles, J. R. (1979) J. Am. Chem. Soc. 101, 510].

Adenosine Triphosphate↗