Kinetic analysis of the action of tissue transglutaminase on peptide and protein substrates.
Tissue transglutaminase (TGase) catalyzes transfer of gamma-acyl moieties of Gln residues in peptides or protein substrates to either water or amine nucleophiles through an acyl-enzyme intermediate formed from initial acyl-transfer to an active site Cys residue. Natural substrates for this enzyme include proteins (e.g., tau, alpha-synuclein, and huntingtin) whose TGase-promoted polymerization may be causative in neurodegenerative diseases. As part of a program to find inhibitors of TGase, we have undertaken kinetic and mechanistic studies of the enzyme from guinea pig (gpTGase) and humans (hTGase). Key findings of this study include: (i) gpTGase-catalyzed transamidation of Z-Gln-Gly by Gly-OMe proceeds essentially as described above but with the involvement of substrate inhibition by Gly-OMe. This phenomena, resulting from the binding of nucleophile to free enzyme, appears to be a common feature of TGase-catalyzed reactions. (ii) Solvent deuterium isotope effects for hydrolysis of Z-Gln-Gly by gpTGase are (D)(k(c)/K(m)) = 0.45 and (D)k(c) = 3.6. While the latter results from general catalysis of deacylation, the former originates purely from the reactant state, hydrogen fractionation factor of the active site thiol with no involvement of general catalysis of acylation. (iii) Studies of the transamidation of N,N-dimethylated casein by Gly-OMe and dansyl-cadaverine suggest a complex kinetic mechanism for both enzymes that reflects contributions from four reactions: Gln hydrolysis, intramolecular transpeptidation, intermolecular transpeptidation, and transamidation by added nucleophile.
Case
April
A
Laboratory for Drug Discovery in Neurodegeneration, Harvard Center for Neurodegeneration and Repair, 65 Landsdowne Street, Fourth Floor, Cambridge, Massachusetts 02139, USA.
Stein
Ross L
RL
D000215:Acylation; D000588:Amines / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002863:Chromogenic Compounds / Q000138:chemical synthesis; D003903:Deuterium / Q000737:chemistry; D005973:Glutamine / Q000737:chemistry; D006168:Guinea Pigs; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008432:Mathematical Computing; D008956:Models, Chemical; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D011503:Transglutaminases / Q000737:chemistry* / Q000378:metabolism*
D000588:Amines; D002863:Chromogenic Compounds; D010446:Peptide Fragments; D005973:Glutamine; D003903:Deuterium; D011503:Transglutaminases