Penicillopepsin-JT2, a Recombinant Enzyme from Penicillium Janthinellum and the Contribution of a Hydrogen Bond in Subsite S3 to Kcat
Protein Science ~2000!, 9:991–1001. Cambridge University Press. Printed in the USA. Copyright © 2000 The Protein Society Penicillopepsin-JT2, a recombinant enzyme from Penicillium janthinellum and the contribution of a hydrogen bond in subsite S3 to kcat QING-NA CAO,1,3 MARLENE STUBBS,1 KENNY Q.P. NGO,1 MICHAEL WARD,2 ANNIE CUNNINGHAM,1 EMIL F. PAI,1 GUANG-CHOU TU,1,3 and THEO HOFMANN1 1 Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada 2 Genencor International, Inc., 925 Page Mill Road, Palo Alto, California 94304-1013 ~Received August 30, 1999; Final Revision February 7, 2000; Accepted March 10, 2000! Abstract The nucleotide sequence of the gene ~ pepA! of a zymogen of an aspartic proteinase from Penicillium janthinellum with a 71% identity in the deduced amino acid sequence to penicillopepsin ~which we propose to call penicillopepsin-JT1! has been determined. The gene consists of 60 codons for a putative leader sequence of 20 amino acid residues, a sequence of about 150 nucleotides that probably codes for an activation peptide and a sequence with two introns that codes for the active aspartic proteinase. This gene, inserted into the expression vector pGPT-pyrG1, was expressed in an aspartic proteinase-free strain of Aspergillus niger var. awamori in high yield as a glycosylated form of the active enzyme that we call penicillopepsin-JT2. After removal of the carbohydrate component with endoglycosidase H, its relative molecular mass is between 33,700 and 34,000. Its kinetic properties, especially the rate-enhancing effects of the presence of alanine residues in positions P3 and P29 of substrates, are similar to those of penicillopepsin-JT1, endothia- pepsin, rhizopuspepsin, and pig pepsin.
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