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Rapid optimization of enzyme substrates using defined substrate mixtures.

Publication ,  Journal Article
Berman, J; Green, M; Sugg, E; Anderegg, R; Millington, DS; Norwood, DL; McGeehan, J; Wiseman, J
Published in: J Biol Chem
January 25, 1992

A strategy is described for the rapid optimization of kcat/Km for protease substrates. Selected positions of a given peptide substrate sequence are varied through synthesis with mixtures of amino acids. Incubation of the resulting peptide mixture with the enzyme of interest and analysis by high pressure liquid chromatography provides a direct measure of analogs with enhanced kcat/Km. High performance liquid chromatography/continuous flow fast atom bombardment mass spectrometry is used to assign structure to each peak in the chromatogram. As an example of the utility and efficiency of "substrate mapping" we describe optimization of the collagenase substrate Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 (where Dnp is dinitrophenyl) at the P'1 and P'2 positions. Six different mixtures were prepared for evaluation, representing the synthesis of 128 different synthetic substrates. "Substrate mapping" has led to Dnp-Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg-NH2, a substrate that possesses a 10-fold better kcat/Km than Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2.

Duke Scholars

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

January 25, 1992

Volume

267

Issue

3

Start / End Page

1434 / 1437

Location

United States

Related Subject Headings

  • Substrate Specificity
  • Spectrometry, Mass, Fast Atom Bombardment
  • Oligopeptides
  • Molecular Sequence Data
  • Microbial Collagenase
  • Mass Spectrometry
  • Kinetics
  • Endopeptidases
  • Chromatography, High Pressure Liquid
  • Biochemistry & Molecular Biology
 

Citation

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Berman, J., Green, M., Sugg, E., Anderegg, R., Millington, D. S., Norwood, D. L., … Wiseman, J. (1992). Rapid optimization of enzyme substrates using defined substrate mixtures. J Biol Chem, 267(3), 1434–1437.
Berman, J., M. Green, E. Sugg, R. Anderegg, D. S. Millington, D. L. Norwood, J. McGeehan, and J. Wiseman. “Rapid optimization of enzyme substrates using defined substrate mixtures.J Biol Chem 267, no. 3 (January 25, 1992): 1434–37.
Berman J, Green M, Sugg E, Anderegg R, Millington DS, Norwood DL, et al. Rapid optimization of enzyme substrates using defined substrate mixtures. J Biol Chem. 1992 Jan 25;267(3):1434–7.
Berman, J., et al. “Rapid optimization of enzyme substrates using defined substrate mixtures.J Biol Chem, vol. 267, no. 3, Jan. 1992, pp. 1434–37.
Berman J, Green M, Sugg E, Anderegg R, Millington DS, Norwood DL, McGeehan J, Wiseman J. Rapid optimization of enzyme substrates using defined substrate mixtures. J Biol Chem. 1992 Jan 25;267(3):1434–1437.

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

January 25, 1992

Volume

267

Issue

3

Start / End Page

1434 / 1437

Location

United States

Related Subject Headings

  • Substrate Specificity
  • Spectrometry, Mass, Fast Atom Bombardment
  • Oligopeptides
  • Molecular Sequence Data
  • Microbial Collagenase
  • Mass Spectrometry
  • Kinetics
  • Endopeptidases
  • Chromatography, High Pressure Liquid
  • Biochemistry & Molecular Biology