Linked Life Data

10965040

Source:http://linkedlifedata.com/resource/pubmed/id/10965040

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
2000-11-29
pubmed:abstractText
The substrate specificity of microbial transglutaminase (MTG) from Streptomyces mobaraensis (formerly categorized Streptoverticillium) was studied using a Streptomyces 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 subtilisin inhibitor, SSI, a homolog of STI2. The results suggest that the P1 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 beta-casein was used as a partner Gln-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 flanking amino acid residues affecting the MTG reactivity of the substrate Lys residue: (i) site-specific mutations from Asn to Lys or Arg at position 69 preceding the amine-donor 70Lys, led to enhanced substrate reactivity; (ii) amino acid replacement at 67Ile with Ser led to higher substrate 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 substrate specificity of guinea pig liver tissue transglutaminase (GTG) was compared with that of MTG using STI2 and its mutants. In contrast to MTG, replacement of Gly by Asp at position 65 was the most favorable for substrate reactivity. Also, 70Lys appeared not to be a prime amine-donor site for GTG-mediated cross-linking, suggesting a difference in substrate recognition between MTG and GTG.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
0021-924X
pubmed:author
pubmed:issnType
Print
pubmed:volume
128
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
415-25
pubmed:dateRevised
2007-12-19
pubmed:meshHeading
pubmed-meshheading:10965040-Amino Acid Sequence, pubmed-meshheading:10965040-Animals, pubmed-meshheading:10965040-Bacterial Proteins, pubmed-meshheading:10965040-Base Sequence, pubmed-meshheading:10965040-Chromatography, pubmed-meshheading:10965040-Cross-Linking Reagents, pubmed-meshheading:10965040-DNA Primers, pubmed-meshheading:10965040-Enzyme-Linked Immunosorbent Assay, pubmed-meshheading:10965040-Genetic Vectors, pubmed-meshheading:10965040-Guinea Pigs, pubmed-meshheading:10965040-Liver, pubmed-meshheading:10965040-Models, Molecular, pubmed-meshheading:10965040-Molecular Sequence Data, pubmed-meshheading:10965040-Mutagenesis, Site-Directed, pubmed-meshheading:10965040-Oxidation-Reduction, pubmed-meshheading:10965040-Polymerase Chain Reaction, pubmed-meshheading:10965040-Rabbits, pubmed-meshheading:10965040-Recombinant Proteins, pubmed-meshheading:10965040-Sequence Alignment, pubmed-meshheading:10965040-Streptomyces, pubmed-meshheading:10965040-Structure-Activity Relationship, pubmed-meshheading:10965040-Substrate Specificity, pubmed-meshheading:10965040-Transglutaminases, pubmed-meshheading:10965040-Trypsin Inhibitors
pubmed:year
2000
pubmed:articleTitle
Substrate specificity analysis of microbial transglutaminase using proteinaceous protease inhibitors as natural model substrates.
pubmed:affiliation
Department of Biological Science and Technology, Science University of Tokyo, Yamazaki, Noda, Chiba 278-8510, Japan. staguchi@postman.riken.go.jp
pubmed:publicationType
Journal Article, Comparative Study, Research Support, Non-U.S. Gov't