A fluorescence-based array screen for transglutaminase substrates.
Transglutaminases (EC 2.3.2.13) form an enzyme family that catalyzes the formation of isopeptide bonds between the γ-carboxamide group of glutamine and the ε-amine group of lysine residues of peptides and proteins. Other primary amines can be accepted in place of lysine. Because of their important physiological and pathophysiological functions, transglutaminases have been studied for 60 years. However, the substrate preferences of this enzyme class remain largely elusive. In this study, we used focused combinatorial libraries of 400 peptides to investigate the influence of the amino acids adjacent to the glutamine and lysine residues on the catalysis of isopeptide bond formation by microbial transglutaminase. Using the peptide microarray technology we found a strong positive influence of hydrophobic and basic amino acids, especially arginine, tyrosine, and leucine. Several tripeptide substrates were synthesized, and enzymatic kinetic parameters were determined both by microarray analysis and in solution.
Institute of Biochemistry and Biotechnology, Department of Enzymology, Project Group gFP5, Martin Luther University Halle-Wittenberg, Weinbergweg 22, 06120 Halle/Saale (Germany).
Miroslav
M
Malešević
Department of Pharmaceutical Technology and Biopharmacy, Institute of Pharmacy, Faculty of Sciences I, Biosciences, Martin Luther University Halle-Wittenberg, Weinbergweg 22, 06120 Halle/Saale (Germany).
Andreas
A
Migge
Department of Pharmaceutical Technology and Biopharmacy, Institute of Pharmacy, Faculty of Sciences I, Biosciences, Martin Luther University Halle-Wittenberg, Weinbergweg 22, 06120 Halle/Saale (Germany).
Thomas C
TC
Hertel
Department of Pharmaceutical Technology and Biopharmacy, Institute of Pharmacy, Faculty of Sciences I, Biosciences, Martin Luther University Halle-Wittenberg, Weinbergweg 22, 06120 Halle/Saale (Germany). markus.pietzsch@pharmazie.uni-halle.de.
Markus
M
Pietzsch
D000595:Amino Acid Sequence; D055162:Biocatalysis; D002482:Cellulose / Q000737:chemistry; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods*; D013050:Spectrometry, Fluorescence; D013302:Streptomyces / Q000201:enzymology; D011503:Transglutaminases / Q000737:chemistry / Q000378:metabolism*
D008567:Membranes, Artificial; D010455:Peptides; D002482:Cellulose; D011503:Transglutaminases
fluorescence; peptides; spot synthesis; substrate specificity; transglutaminase