THE AMINE-DONOR SUBSTRATE-SPECIFICITY OF TISSUE-TYPE TRANSGLUTAMINASE- INFLUENCE OF AMINO-ACID-RESIDUES FLANKING THE AMINE-DONOR LYSINE RESIDUE

The amine-donor substrate specificity of tissue-type transglutaminase has been studied in a series of recombinant alpha A-crystallin mutants . These mutant proteins have been provided with a potential substrate lysine residue, flanked by different amino acid residues, in the C-ter minal extended arm of alpha A-crystallin. A biotinylated amine-accepto r hexapeptide was used as a probe for labelling the amine-donor sites. Wild-type bovine alpha A-crystallin does not function as an amine-don or substrate for tissue-type transglutaminase. Yet, upon introduction of a lysine residue at the C-terminal or penultimate position, all mut ant alpha A-crystallins act as amine-donor substrates, although to dif ferent extents. This shows that accessibility is the primary requireme nt for a lysine residue to function as an amine-donor substrate for tr ansglutaminase and that the enzyme has a broad tolerance towards the n eighbouring residues. However, the nature of the flanking amino acid r esidues does clearly affect the reactivity of the substrate lysine res idue. Notably, we found that a proline or glycine residue in front of the substrate lysine has a strong adverse effect on the substrate reac tivity as compared to a preceding leucine, serine, alanine or arginine residue.