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Kinetic studies of papain: Effect of P3′ substituents and donor/acceptor pairs of intramolecularly quenched fluorogenic substrates

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Summary

Two series of intramolecularly quenched fluorogenic oligopeptide substrates with the general sequences DABCYL-Lys-Phe-Gly-Gly-Ala-Xxx-EDANS and Abz-Lys-Phe-Gly-Gly-Ala-Xxx-Tyr(m-NO2)-NH2 have been used to explore the effect of P3′ substituents and donor/acceptor pairs on the kinetic parameters for papain-catalyzed hydrolysis. The steady-state constants are moderately affected by amino acid or fluorophore/quencher replacement. No correlation between the hydrophobicity of the P3′ substituent and the kinetic parameters was found.

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Abbreviations

Abz:

anthranilic acid or anthranilamide

Boc:

tert-butyloxycarbonyl

BOP:

benzotriazolyloxy tris-(dimethylamino)-phosphonium hexafluorophosphate

DABCYL:

4-(4-dimethylaminophenylazo)benzoic acid

DCM:

dichloro-methane

DIEA:

diisopropylethylamine

DIPCDI:

diisopropylcarbodiimide

DMF:

N,N-dimethylformamide

DMSO:

dimethylsulfoxide

E-64:

trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane

EDCI:

1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide hydrochloride

EDTA:

ethylenediaminetetraacetic acid

EDANS:

5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid

FABMS:

fast atom bombardment mass spectrometry

Fmoc:

9-fluorenylmethyloxycarbonyl

HOBt:

1-hydroxybenzotriazole

HPLC:

high pressure liquid chromatography

MBHA:

4-methylbenzhydrylamine (resin)

MCA:

methylcoumarylamide

Nle:

norleucine

PAL:

tris(alkoxy)benzylamide linker

Su:

succinimide

TFA:

trifluoroacetic acid

tR :

retention time

Tyr(m-NO2):

meta-nitrotyrosine

Z:

benzyloxycarbonyl

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Author notes

  1. Carlos García-Echeverría

    Present address: Pharmaceuticals Division, Ciba-Geigy Ltd., CH-4002, Basel, Switzerland

Authors and Affiliations

  1. School of Pharmacy, University of Wisconsin-Madison, 425 North Charter Street, 53706, Madison, WI, U.S.A.

    Carlos García-Echeverría & Daniel H. Rich

  2. Department of Chemistry, University of Wisconsin-Madison, 425 North Charter Street, 53706, Madison, WI, U.S.A.

    Carlos García-Echeverría & Daniel H. Rich

Authors
  1. Carlos García-Echeverría
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  2. Daniel H. Rich
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Additional information

Abbreviations used for amino acids follow the recommendations of the IUPAC-IUB Commission of Biochemical Nomenclature [Eur. J. Biochem., 138 (1984) 9]. Amino acid symbols denote the l-configuration.

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García-Echeverría, C., Rich, D.H. Kinetic studies of papain: Effect of P3′ substituents and donor/acceptor pairs of intramolecularly quenched fluorogenic substrates. Lett Pept Sci 2, 77–82 (1995). https://doi.org/10.1007/BF00128501

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  • DOI: https://doi.org/10.1007/BF00128501

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