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Mechanism of Action of Guinea Pig Liver Transglutaminase

Folk, J.E. ; Gross, Michael

The Journal of biological chemistry, 1971-11, Vol.246 (21), p.6683-6691 [Periódico revisado por pares]

American Society for Biochemistry and Molecular Biology

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  • Título:
    Mechanism of Action of Guinea Pig Liver Transglutaminase
  • Autor: Folk, J.E. ; Gross, Michael
  • É parte de: The Journal of biological chemistry, 1971-11, Vol.246 (21), p.6683-6691
  • Descrição: The reaction of α-bromo-4-hydroxy-3-nitroacetophenone (BHNA) with transglutaminase in the presence of CaCl 2 (25 m m ) produces a catalytically inactive labeled protein in which the phenacyl group is covalently attached to the active site —SH. The spectral properties of this group attached to the enzyme are consistent with that of the group in a hydrophobic region of the molecule. Addition of ethylenediaminetetraacetate results in a shift of the spectrum toward shorter wave lengths, indicative of a more polar environment for this —SH in the absence of Ca ++ . Attachment of the phenacyl group to positions in the enzyme other than the active —SH by reaction with BHNA in the absence of Ca ++ results in losses in transferase activity, but essentially no loss in esterase activity. The spectrum of the groups bound to enzyme in the absence of Ca ++ is identical with that of the phenacyl group in water. This spectrum is unchanged by subsequent addition of Ca ++ . In the catalytically inactive forms of transglutaminase, produced by the reaction of d and l forms of methyl N -(2-hydroxy-5-nitrophenylacetyl)-2-amino-4-oxo-5-chloropentanoate (PACK) and 1-chloro-4-(2-hydroxy-5-nitrophenylacetyl)amidobutan-2-one (PBCK) with enzyme in the presence of Ca ++ , the phenolic reporter group is attached covalently to the enzyme's active —SH. The rapid rate of inactivation by l -PACK compared to d -PACK and PBCK implies that l -PACK, by virtue of its structural similarity to transglutaminase substrates, is properly oriented at the substrate-binding site of enzyme prior to the covalent reaction. The pK a of the phenolic group in the acyl protion of each of these inactivators is shifted towards that of a weaker acid in the reporter-labeled enzyme proteins. The identical changes in pK a with each inactivator suggest that the phenylacetyl side chain in each case is positioned in the same manner within the matrix of the calcium-activated enzyme derivative. Addition of EDTA results in a shift in pK a of the phenolic group in each enzyme derivative back to that of the parent inactivator. This, together with the findings with BHNA, forms the basis for a suggestion that the active —SH is located at or near the surface in the unactivated enzyme, i.e. , in the absence of Ca ++ . Active site titration procedures for transglutaminase are described. A rate assay procedure utilizing either BHNA or l -PACK was found to give results in excellent agreement with those of a direct spectrophotometric method carried out with the use of BHNA. The latter titration is based on the differences in the absorption spectrum of the phenacyl group bound to the enzyme's active —SH and that of this group attached to other positions on the enzyme molecule.
  • Editor: American Society for Biochemistry and Molecular Biology
  • Idioma: Inglês
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  • Realização: ABCD-USP USP   Logos de Redes Sociais: Freepik

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