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
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.