Penicillopepsin-JT2, a recombinant enzyme from Penicillium janthinellum and the contribution of a hydrogen bond in subsite S-3 to k(cat)

Citation
Qn. Cao et al., Penicillopepsin-JT2, a recombinant enzyme from Penicillium janthinellum and the contribution of a hydrogen bond in subsite S-3 to k(cat), PROTEIN SCI, 9(5), 2000, pp. 991-1001
Citations number
59
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
PROTEIN SCIENCE
ISSN journal
0961-8368 → ACNP
Volume
9
Issue
5
Year of publication
2000
Pages
991 - 1001
Database
ISI
SICI code
0961-8368(200005)9:5<991:PAREFP>2.0.ZU;2-C
Abstract
The nucleotide sequence of the gene (pepA) of a zymogen of an aspartic prot einase from Penicillium janthinellum with a 71% identity in the deduced ami no acid sequence to penicillopepsin (which we propose to call penicillopeps in-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 nucleoti des that probably codes for an activation peptide and a sequence with two i ntrons that codes for the active aspartic proteinase. This gene, inserted i nto the expression vector pGPT-pyrG1, was expressed in an aspartic proteina se-free strain of Aspergillus niger var. awamori in high yield as a glycosy lated form of the active enzyme that we call penicillopepsin-JT2. After rem oval of the carbohydrate component with endoglycosidase H, its relative mol ecular mass is between 33,700 and 34,000. Its kinetic properties, especiall y the rate-enhancing effects of the presence of alanine residues in positio ns P-3 and P-2' of substrates, are similar to those of penicillopepsin-JT1, endothiapepsin, rhizopuspepsin, and pig pepsin. Earlier findings suggested that this rate-enhancing effect was due to a hydrogen bond between the -NH - of P-3 and the hydrogen bond accepting oxygen of the side chain of the fo urth amino acid residue C-terminal to Asp215. Thr219 of penicillopepsin-JT2 was mutated to Ser, Val, Gly, and Ala. Thr219Ser showed an increase in k(c at) when a P-3 residue was present in the substrate, which was similar to t hat of the wild-type, whereas the mutants Thr219Val, Thr219Gly, and Thr219A la showed no significant increase when a P-3 residue was added. The results show that the putative hydrogen bond alone is responsible for the increase . We propose that by locking the -NH- of P-3 to the enzyme, the scissile pe ptide bond between P-1 and P-1' becomes distorted toward a tetrahedral conf ormation and becomes more susceptible to nucleophilic attack by the catalyt ic apparatus without the need of a conformational change in the enzyme.