Structural and functional integrity of specificity and catalytic sites of trypsin

LÁSZLÓ GRÁF, GYÖRGY HEGYI, ISTVÁN LIKÓ, JÓZSEF HEPP, KÁLMÁN MEDZIHRADSZKY, CHARLES S. CRAIK, WILLIAM J. RUTTER

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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 Km 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
DOIs
Publication statusPublished - Dec 1988

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Keywords

  • aspartic acid residue
  • catalytic site
  • glutamic acid residue
  • trypsin

ASJC Scopus subject areas

  • Biochemistry

Cite this

GRÁF, LÁSZLÓ., HEGYI, GYÖRGY., LIKÓ, ISTVÁN., HEPP, JÓZSEF., MEDZIHRADSZKY, KÁLMÁN., CRAIK, CHARLES. S., & RUTTER, WILLIAM. J. (1988). Structural and functional integrity of specificity and catalytic sites of trypsin. International journal of peptide and protein research, 32(6), 512-518. https://doi.org/10.1111/j.1399-3011.1988.tb01382.x