Substrate Specificity Analysis of Microbial Transglutaminase Using Proteinaceous Protease Inhibitors As Natural Model Substrates1

The substrate specificity of microbial transglutaminase (MTG) from Streptomyces mobaraensis (formerly categorized Streptoverticillium) was studied using a Streptomy-ces proteinaceous protease inhibitor, STI2, as a model amine-donor substrate. Chemical modification and mutational analysis to address the substrate requirements for MTG were carried out around the putative reactive site region of STI2 on the basis of the highly refined tertiary structure and the solvent accessibility index of Streptomyces sub-tilisin inhibitor, SSI, a homolog of STT2. The results suggest that the PI reactive center site (position 70 of STI2) for protease subtilisin BPN ' or trypsin may be the prime Lys residue that can be recognized by MTG, when succinylated P-casein was used as a part-ner Gin-substrate. It is characteristic in that the same primary enzyme contact region of STI2 is shared by both enzymes, MTG and proteases. For quantitative analysis of the TG reaction, we established an ELISA-based monitoring assay system using an anti-SSI polyclonal antibody highly cross-reactive with STI2. Site-specific STI2 mutants were prepared by an Escherichia coli expression-secretion vector system and subjected to the assay system. We reached several conclusions concerning the nature of the flankin