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Journal of Biochemistry Advance Access published online on October 9, 2009
Journal of Biochemistry, doi:10.1093/jb/mvp158
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© The authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Differences in the P1' Substrate Specificities of Pepsin A and Chymosin

Hakuto Kageyama1,#,*, Hiroshi Ueda2, Takafumi Tezuka1, Asami Ogasawara3, Yuichi Narita3,#, Takashi Kageyama3 and Masao Ichinose4

1School of science, Biological science, Nagoya University; 2 AJINOMOTO CO., INC.; 3Primate Research Institute, Kyoto University; and 4Department of Gastroenterology, School of Medicine, Wakayama Medical University

*Correspondence to Hakuto Kageyama, Tel: +81-59-346-0121, Fax: +81-59-346-0127, E-mail: hakuto_kageyama{at}ajinomoto.com

Received August 22, 2009; Accepted September 13, 2009


  Abstract

Porcine pepsin A and bovine chymosin are typical models of aspartic proteinases. The hydrolytic specificities of these proteinases, along with those of human pepsin A and monkey chymosin, were investigated with 29 peptide substrates that included various P1' variants of seven parent peptides. From these peptides, AFPLEF{downarrow}FREL was preferred by pepsin A and chymosin, while its P1' variant, AFPLEF{downarrow}EREL, was preferred by bovine chymosin. Porcine and human pepsin A showed similar hydrolytic specificities, strongly preferring a hydrophobic/aromatic residue at P1' of any type of peptide. This specificity is well explained by the very hydrophobic nature of the S1' subsite that consists of Tyr189, Ile213, Ile300, Met289, Val/Leu291, and Leu298. The first three residues are well conserved in pepsin family enzymes. Although bovine and monkey chymosin showed similar P1' specificity, bovine chymosin preferred peptides having Glu at P1', while monkey chymosin preferred peptides having Lys at P1'. The dual characteristics of chymosin are due to the occurrence of polar/charged residues in the S1' subsite, such as Glu/Asp289, Gln298, and Lys/Gln299, which are different from the S1' subsite of pepsin A. Molecular models suggest that Glu in position 289 of bovine chymosin, and Asp in position 289 of monkey chymosin are responsible for the difference in P1' specificities between the chymosins.

Key Words: pepsin A, chymosin, aspartic proteinase, proteolytic activity, substrate specificity

#Present address: Fine Technomogy Dept., Fine Chemical & Pharmaceutical Industrialization Center, Amino Acids Company, AJINOMOTO CO., INC. (H. K.); Friedrich Miescher Institute,CH-4058 Basel, Switzerland (Y. N.)


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