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Site-directed mutagenesis shows that tyrosine 248 of carboxypeptidase A does not play a crucial role in catalysis

Nature volume 317, pages 551–555 (1985)Cite this article

Abstract

The residue Tyr 248 of carboxypeptidase A (CPA) is thought to play a role in catalysis by contributing a proton to the incipient amine anion generated during cleavage of peptide substrates1. To test this hypothesis we have modified the rat CPA cDNA2 by site-directed mutagenesis3 so that the codon for Tyr 248 is replaced by that for Phe. Here, we report the expression of the cDNAs for proCPA and its Tyr-to-Phe variant in yeast via the α-factor system4–6. Following zymogen activation by trypsin, wild-type CPA (CPA-WT) and variant CPA (CPA-Phe 248) were purified to homogeneity and characterized enzymatically. CPA-Phe 248 displays essentially undiminished values for the catalytic constant (kcat) towards various peptide and ester substrates. However, the Michaelis constants (Km values) of peptide substrates and the inhibition constant (Ki) of the potato carboxypeptidase inhibitor7 are increased 6-fold and 70-fold, respectively. These data suggest that the phenolic hydroxyl of Tyr 248 does not act as the requisite general acid catalyst but participates in ligand binding.

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Author notes

  1. Donald Hilvert: Laboratory of Bio-organic Chemistry and Biochemistry, Rockefeller University, New York, New York 10021, USA.

  2. Mickey S. Urdea: Chiron Corporation, 4560 Horton Street, Emeryville, California, USA.

Authors and Affiliations

  1. Hormone Research Laboratory and Department of Biochemistry and Biophysics, University of California, San Francisco, California, 94143, USA

    Stephen J. Gardell, Charles S. Craik, Donald Hilvert, Mickey S. Urdea & William J. Rutter

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  1. Stephen J. Gardell
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  2. Charles S. Craik
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  3. Donald Hilvert
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  4. Mickey S. Urdea
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  5. William J. Rutter
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Gardell, S., Craik, C., Hilvert, D. et al. Site-directed mutagenesis shows that tyrosine 248 of carboxypeptidase A does not play a crucial role in catalysis. Nature 317, 551–555 (1985). https://doi.org/10.1038/317551a0

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