Chemical modification of the structures and functions of proteins by the cofactor reconstitution method

Itaru Hamachi, Seiji Shinkai

Research output: Contribution to journalReview articlepeer-review

50 Citations (Scopus)


A new strategy for the semi-synthesis of proteins and enzymes bearing non-natural functional groups is described. In line with our proposal that the replacement of native cofactors by modified cofactors may represent a useful and general methodology for introducing non-natural molecules into cofactor-dependent proteins and enzymes, we have shown that semi-synthetic cofactors bearing non-natural functional units can be incorporated in the proximity of the active sites of enzymes by cofactor reconstitution. A variety of non-natural building units such as a hydrophobic alkyl chain, a polyanion cluster, metal complexes, electron donors or acceptors, a photochromic moiety, peptides, and artificial receptors have successfully been introduced into hemoproteins (myoglobin, hemoglobin, and cytochrome b562) and their effects on the holoproteins have been examined. Incorporation of a long alkyl chain, for example, greatly facilitates conversion of a water-soluble protein into a membrane-bound protein. Attachment of a photosensitizer has enabled us to switch the enzyme activity by means of visible light. Artificial receptors introduced in the proximity of the active site can modulate the structures and activities of native proteins in response to specific guest molecules. This concept has been expanded to include flavoenzymes such as glucose oxidase. An immense variety of non- natural molecules are available that are potentially useful for protein engineering. By combination of the present cofactor reconstitution method with other techniques such as site-directed mutagenesis, chemical modification, peptide semi-synthesis, and non-natural amino acid incorporation using suppressor t-RNA, it should be possible to artificially manipulate native protein molecules with a similar degree of flexibility as that with which organic chemists manipulate small molecules.

Original languageEnglish
Pages (from-to)539-549
Number of pages11
JournalEuropean Journal of Organic Chemistry
Issue number3
Publication statusPublished - Jan 1 1999

All Science Journal Classification (ASJC) codes

  • Physical and Theoretical Chemistry
  • Organic Chemistry


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