Penicillopepsin-JT2, a recombinant enzyme from Penicillium janthinellum and the contribution of a hydrogen bond in subsite S3 to k(cat).
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.
Cao
Q N
QN
Department of Biochemistry, University of Toronto, Ontario, Canada.
Stubbs
M
M
Ngo
K Q
KQ
Ward
M
M
Cunningham
A
A
Pai
E F
EF
Tu
G C
GC
Hofmann
T
T
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000235:genetics; D001230:Aspergillus / Q000201:enzymology; D001426:Bacterial Proteins; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D043384:Glutamyl Aminopeptidase; D006031:Glycosylation; D006860:Hydrogen Bonding; D007438:Introns; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine
D001426:Bacterial Proteins; D003062:Codon; D011994:Recombinant Proteins; D000626:Aminopeptidases; D043384:Glutamyl Aminopeptidase; C022325:aspartic proteinase A; D016282:Aspartic Acid Endopeptidases