SUBSTRATE REQUIREMENTS FOR TRANSGLUTAMINASES - INFLUENCE OF THE AMINO-ACID RESIDUE PRECEDING THE AMINE DONOR LYSINE IN A NATIVE PROTEIN

Thirteen recombinant alpha A-crystallin mutants were constructed that differed in the type of amino acid residue directly preceding the sole amine donor lysine for transglutaminases in this protein. The capacit y of these mutants to be cross-linked to amine acceptor substrates by tissue transglutaminase and factor XIII was assessed. Two different bi otinylated glutamine-containing oligopeptides were used as amine accep tor probes. It appears that the type of residue preceding the amine do nor lysine has a considerable influence on the substrate potential of alpha A-crystallin for transglutaminases. This influence shows qualita tively similar trends for tissue transglutaminase and factor XIII and is irrespective of the amine acceptor probe. In general, glycine or as partic acid before the amine donor lysine has the strongest adverse ef fects on substrate reactivity, and proline, histidine, and tryptophan are less favorable. Valine, arginine, and phenylalanine, and to a more variable or somewhat lesser extent also serine, alanine, leucine, tyr osine, and asparagine, have an enhancing effect. This pattern of prefe rence is largely in agreement with that observed for the limited numbe r of characterized amine donor lysines in protein substrates for trans glutaminases. It can be concluded that tissue transglutaminase and fac tor XIII have a rather broad yet clearly differentiated tolerance with respect to the residue preceding the amine donor lysine substrate in native proteins.