Substrate specificity analysis of microbial transglutaminase using proteinaceous protease inhibitors as natural model substrates

The substrate specificity of microbial transglutaminase (MTG) from Streptom yces mobaraensis (formerly categorized Streptoverticillium) was studied usi ng a Streptomyces proteinaceous protease inhibitor, STI2, as a model amine- donor substrate. Chemical modification and mutational analysis to address t he substrate requirements for MTG were carried out around the putative reac tive site region of STI2 on the basis of the highly refined tertiary struct ure and the solvent accessibility index of Streptomyces subtilisin inhibito r, SSI, a homolog of STI2, The results suggest that the pi reactive center site (position 70 of STI2) for protease subtilisin BPN' or trypsin may be t he prime Lys residue that can be recognized by MTG, when succinylated beta- casein. was used as a partner Gin-substrate. It is characteristic in that t he same primary enzyme contact region of STI2 is shared by both enzymes, MT G and proteases, For quantitative analysis of the TG reaction, we establish ed an ELISA-based monitoring assay system using an anti-SSI polyclonal anti body highly cross-reactive with STI2, Site-specific STI2 mutants were prepa red by an Escherichia coli expression-secretion vector system and subjected to the assay system. We reached several conclusions concerning the nature of the flanking amino acid residues affecting the MTG reactivity of the sub strate Lys residue: (i) site-specific mutations from Asn to Lys or Arg at p osition 69 preceding the amine-donor 70Lys, led to enhanced substrate react ivity; (ii) amino acid replacement at 67Ile with Ser led to higher substrat e reactivity, (iii) additive effects were obtained by a combination of the positive mutations at positions 67 and 69 as described above, and (iv) Gly at position 65 might be essential for MTG; reaction. Moreover, the substrat e specificity of guinea pig liver tissue transglutaminase (GTG) was compare d with that of MTG using STI2 and its mutants, In contrast to MTG, replacem ent of Gly by Asp at position 65 was the most favorable for substrate react ivity. Also,70Lys appeared not to be a prime amine-donor site for GTG-media ted cross-linking, suggesting a difference in substrate recognition between . MTG and GTG.