Electrostatic complementarity within the substrate-binding pocket of trypsin.

L. Gráf, A. Jancsó, L. Szilágyi, G. Hegyi, K. Pintér, G. Náray-Szabó, J. Hepp, K. Medzihradszky, W. J. Rutter

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Abstract

The aspartic residue (Asp-189) at the base of the substrate-binding pocket of trypsin was replaced by serine (present in a similar position in chymotrypsin) through site-directed mutagenesis. The wild-type (with Asp-189 in the mature trypsin sequence) and mutant (Ser-189) trypsinogens were expressed in Escherichia coli, purified to homogeneity, activated by enterokinase, and tested with a series of fluorogenic tetrapeptide substrates with the general formula succinyl-Ala-Ala-Pro-Xaa-AMC, where AMC is 7-amino-4-methyl-coumarin and Xaa is Lys, Arg, Tyr, Phe, Leu, or Trp. As compared to [Asp189]trypsin, the activity of [Ser189]trypsin on lysyl and arginyl substrates decreased by about 5 orders of magnitude while its Km values increased only 2- to 6-fold. In contrast, [Ser189]trypsin was 10-50 times more active on the less preferred, chymotrypsin-type substrates (tyrosyl, phenylalanyl, leucyl, and tryptophanyl). The activity of [Ser189]trypsin on lysyl substrate was about 100-fold greater at pH 10.5 than at pH 7.0, indicating that the unprotonated lysine is preferred. Assuming the reaction mechanisms of the wild-type and mutant enzymes to be the same, we calculated the changes in the transition-state energies for various enzyme-substrate pairs to reflect electrostatic and hydrogen-bond interactions. The relative binding energies (E) in the transition state are as follows: EII greater than EPP greater than EPA greater than EIP approximately equal to EIA, where I = ionic, P = nonionic but polar, and A = apolar residues in the binding pocket. These side-chain interactions become prominent during the transition of the Michaelis complex to the tetrahedral transition-state complex.

Original languageEnglish
Pages (from-to)4961-4965
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume85
Issue number14
Publication statusPublished - Jul 1988

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Static Electricity
Trypsin
Chymotrypsin
Enteropeptidase
Trypsinogen
Enzymes
Site-Directed Mutagenesis
Fluorescent Dyes
Serine
Lysine
Hydrogen
Escherichia coli

ASJC Scopus subject areas

  • General
  • Genetics

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Electrostatic complementarity within the substrate-binding pocket of trypsin. / Gráf, L.; Jancsó, A.; Szilágyi, L.; Hegyi, G.; Pintér, K.; Náray-Szabó, G.; Hepp, J.; Medzihradszky, K.; Rutter, W. J.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 85, No. 14, 07.1988, p. 4961-4965.

Research output: Contribution to journalArticle

Gráf, L, Jancsó, A, Szilágyi, L, Hegyi, G, Pintér, K, Náray-Szabó, G, Hepp, J, Medzihradszky, K & Rutter, WJ 1988, 'Electrostatic complementarity within the substrate-binding pocket of trypsin.', Proceedings of the National Academy of Sciences of the United States of America, vol. 85, no. 14, pp. 4961-4965.
Gráf, L. ; Jancsó, A. ; Szilágyi, L. ; Hegyi, G. ; Pintér, K. ; Náray-Szabó, G. ; Hepp, J. ; Medzihradszky, K. ; Rutter, W. J. / Electrostatic complementarity within the substrate-binding pocket of trypsin. In: Proceedings of the National Academy of Sciences of the United States of America. 1988 ; Vol. 85, No. 14. pp. 4961-4965.
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abstract = "The aspartic residue (Asp-189) at the base of the substrate-binding pocket of trypsin was replaced by serine (present in a similar position in chymotrypsin) through site-directed mutagenesis. The wild-type (with Asp-189 in the mature trypsin sequence) and mutant (Ser-189) trypsinogens were expressed in Escherichia coli, purified to homogeneity, activated by enterokinase, and tested with a series of fluorogenic tetrapeptide substrates with the general formula succinyl-Ala-Ala-Pro-Xaa-AMC, where AMC is 7-amino-4-methyl-coumarin and Xaa is Lys, Arg, Tyr, Phe, Leu, or Trp. As compared to [Asp189]trypsin, the activity of [Ser189]trypsin on lysyl and arginyl substrates decreased by about 5 orders of magnitude while its Km values increased only 2- to 6-fold. In contrast, [Ser189]trypsin was 10-50 times more active on the less preferred, chymotrypsin-type substrates (tyrosyl, phenylalanyl, leucyl, and tryptophanyl). The activity of [Ser189]trypsin on lysyl substrate was about 100-fold greater at pH 10.5 than at pH 7.0, indicating that the unprotonated lysine is preferred. Assuming the reaction mechanisms of the wild-type and mutant enzymes to be the same, we calculated the changes in the transition-state energies for various enzyme-substrate pairs to reflect electrostatic and hydrogen-bond interactions. The relative binding energies (E) in the transition state are as follows: EII greater than EPP greater than EPA greater than EIP approximately equal to EIA, where I = ionic, P = nonionic but polar, and A = apolar residues in the binding pocket. These side-chain interactions become prominent during the transition of the Michaelis complex to the tetrahedral transition-state complex.",
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AU - Gráf, L.

AU - Jancsó, A.

AU - Szilágyi, L.

AU - Hegyi, G.

AU - Pintér, K.

AU - Náray-Szabó, G.

AU - Hepp, J.

AU - Medzihradszky, K.

AU - Rutter, W. J.

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N2 - The aspartic residue (Asp-189) at the base of the substrate-binding pocket of trypsin was replaced by serine (present in a similar position in chymotrypsin) through site-directed mutagenesis. The wild-type (with Asp-189 in the mature trypsin sequence) and mutant (Ser-189) trypsinogens were expressed in Escherichia coli, purified to homogeneity, activated by enterokinase, and tested with a series of fluorogenic tetrapeptide substrates with the general formula succinyl-Ala-Ala-Pro-Xaa-AMC, where AMC is 7-amino-4-methyl-coumarin and Xaa is Lys, Arg, Tyr, Phe, Leu, or Trp. As compared to [Asp189]trypsin, the activity of [Ser189]trypsin on lysyl and arginyl substrates decreased by about 5 orders of magnitude while its Km values increased only 2- to 6-fold. In contrast, [Ser189]trypsin was 10-50 times more active on the less preferred, chymotrypsin-type substrates (tyrosyl, phenylalanyl, leucyl, and tryptophanyl). The activity of [Ser189]trypsin on lysyl substrate was about 100-fold greater at pH 10.5 than at pH 7.0, indicating that the unprotonated lysine is preferred. Assuming the reaction mechanisms of the wild-type and mutant enzymes to be the same, we calculated the changes in the transition-state energies for various enzyme-substrate pairs to reflect electrostatic and hydrogen-bond interactions. The relative binding energies (E) in the transition state are as follows: EII greater than EPP greater than EPA greater than EIP approximately equal to EIA, where I = ionic, P = nonionic but polar, and A = apolar residues in the binding pocket. These side-chain interactions become prominent during the transition of the Michaelis complex to the tetrahedral transition-state complex.

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