Penicillopepsin-JT2, a recombinant enzyme from Penicillium janthinellum and the contribution of a hydrogen bond in subsite S3 to k(cat)

• Published in: Protein science 2000-05, Vol.9 (5), p.991-1001
• Main Authors: Cao, Q N, Stubbs, M, Ngo, K Q, Ward, M, Cunningham, A, Pai, E F, Tu, G C, Hofmann, T
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Aminopeptidases - genetics; Aminopeptidases - metabolism; Animals; Aspartic Acid Endopeptidases - chemistry; Aspartic Acid Endopeptidases - genetics; Aspergillus - enzymology; Bacterial Proteins; Base Sequence; Blotting, Southern; Cloning, Molecular; Codon; Escherichia coli - metabolism; Gene Library; Glutamyl Aminopeptidase; Glycosylation; Hydrogen Bonding; Introns; Kinetics; Models, Genetic; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Conformation; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Restriction Mapping; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Swine
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.9.5.991

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 P2' 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 P3 and the hydrogen bond accepting oxygen of the side chain of the fourth 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(cat) when a P3 residue was present in the substrate, which was similar to that of the wild-type, whereas the mutants Thr219Val, Thr219Gly, and Thr219Ala showed no significant increase when a P3 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 P3 to the enzyme, the scissile peptide bond between P1 and P1' becomes distorted toward a tetrahedral conformation and becomes more susceptible to nucleophilic attack by the catalytic apparatus without the need of a conformational change in the enzyme.