Structural and functional integrity of specificity and catalytic sites of trypsin

L. Gráf, G. Hegyi, I. Liko, J. Hepp, K. Medzihradszky, C. S. Craik, W. J. Rutter

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

The aspartic acid residue at the bottom of the substrate-binding pocket of trypsin was replaced by glutamic acid through site-directed mutagenesis. The wild-type (Asp-189) and mutant (Glu-189) trypsinogens were expressed in E. coli, purified to homogeneity activated by enterokinase, and tested on a series of fluorogenic tetrapeptide substrates. The substrates were of the general formula succinyl-Ala-Ala-Pro-X-AMC, where AMC is 7-amino-4-methylcoumarin and X is Lys, Arg, or Orn (ornithine). As compared to Asp-189 trypsin, the activity of Glu-189 trypsin on lysyl and arginyl substrates decreased by 3-4 orders of magnitude while its K(m) values did not significantly change. Lengthening the side-chain of Asp-189 by one methylene group could not be compensated for by shortening the side-chain of the substrate, since Glu-189 trypsin had no measurable activity on the ornithyl substrate. The replacement of Asp-189 with glutamic acid at the base of the substrate-binding pocket of trypsin appears to distort the structure of the critical transition-state complex. This could happen by disrupting interactions normally associated with Asp-189, and by altering the relative position of the scissile peptide bond in the active site of the enzyme.

Original languageEnglish
Pages (from-to)512-518
Number of pages7
JournalInternational Journal of Peptide and Protein Research
Volume32
Issue number6
Publication statusPublished - 1988

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Trypsin
Catalytic Domain
Substrates
Glutamic Acid
Enteropeptidase
Trypsinogen
Ornithine
Site-Directed Mutagenesis
Fluorescent Dyes
Aspartic Acid
Mutagenesis
Escherichia coli
Peptides
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Gráf, L., Hegyi, G., Liko, I., Hepp, J., Medzihradszky, K., Craik, C. S., & Rutter, W. J. (1988). Structural and functional integrity of specificity and catalytic sites of trypsin. International Journal of Peptide and Protein Research, 32(6), 512-518.

Structural and functional integrity of specificity and catalytic sites of trypsin. / Gráf, L.; Hegyi, G.; Liko, I.; Hepp, J.; Medzihradszky, K.; Craik, C. S.; Rutter, W. J.

In: International Journal of Peptide and Protein Research, Vol. 32, No. 6, 1988, p. 512-518.

Research output: Contribution to journalArticle

Gráf, L, Hegyi, G, Liko, I, Hepp, J, Medzihradszky, K, Craik, CS & Rutter, WJ 1988, 'Structural and functional integrity of specificity and catalytic sites of trypsin', International Journal of Peptide and Protein Research, vol. 32, no. 6, pp. 512-518.
Gráf, L. ; Hegyi, G. ; Liko, I. ; Hepp, J. ; Medzihradszky, K. ; Craik, C. S. ; Rutter, W. J. / Structural and functional integrity of specificity and catalytic sites of trypsin. In: International Journal of Peptide and Protein Research. 1988 ; Vol. 32, No. 6. pp. 512-518.
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