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. 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 kcat 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 P19 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. Keywords: aspartic proteinases; penicillopepsin; penicillopepsin gene; subsite S3 effects Aspartic proteinases are a family of proteolytic enzymes charac- least seven amino acids of a substrate molecule ~Tang et al., 1978!. terized by two essential aspartic acid residues at the active site. It has been known for many years that the catalytic efficiency—as These are located in an extended cleft that can accommodate at expressed by kcat—of the aspartic proteinases pepsin, cathepsin D, and rhizopuspepsin is strongly dependent on the chain length of a substrate peptide ~Sampath-Kumar & Fruton, 1974; Fruton, 1976!, Reprint requests to: Theo Hofmann, Department of Biochemistry, Uni- versity of Toronto, Toronto, Ontario M5S 1A8, Canada; e-mail: theo@ whereas the apparent binding—as expressed by Km—is affected hera.med.utoronto.ca. only little by the substrate chain length. Subsequently, we obtained 3 Present address: Department of Pathology, Anatomy and Cell Biology, evidence, first with penicillopepsin-JT1 ~Hofmann et al., 1988! Thomas Jefferson University, Philadelphia, Pennsylvania 19107. and later with endothiapepsin, rhizopuspepsin, and pig pepsin Abbreviations: A., Aspergillus; CD, circular dichroism; FPLC, fast ~Balbaa et al., 1993!, that two positions in the substrate, namely P* performance liquid chromatography; endo H, endoglycosidase H; Nph, 3 p-nitrophenylalanyl; P., Penicillium; PCR, polymerase chain reaction; PTH, and P29, were largely responsible for the increases in kcat. These phenyl thiohydantoin. The definition of Schecter and Berger ~1967! for increases ranged from 31- to 50-fold for each position with denoting amino acid residues in peptide substrates as P1 to Pn and P19 to Pn9, penicillopepsin-JT1 ~Hofmann et al., 1988!, up to 100-fold with and subsites in the enzyme to which the side chains of these residues bind endothiapepsin and 40-fold to .300-fold for rhizopuspepsin as S1 to Sn and S19 to Sn9 is used throughout. The amino acids in the deduced sequence of penicillopepsin-JT2 are numbered sequentially, starting from ~Balbaa et al., 1993!, whereas residues in positions P2, P4, P39, and the first activation site ~Fig. 2!; the corresponding pepsin numbers are in P49 caused increases that ranged from 0 to only about five fold with parentheses. the three fungal enzymes ~Hofmann et al., 1988; Balbaa et al., 991 992 Q. Cao et al. 1993!. With pepsin the largest increases were also observed in richia coli because in the latter the zymogen is produced in relatively positions P3 and P29 where they were as high as 1,380-fold ~Balbaa low yield in inclusion bodies. This product has to be refolded after et al., 1993!. extraction and activated to give the active proteinase. The charac- We suggested previously that a hydrogen bond that forms be- terization of the active enzyme as well as the construction and tween the -NH- of P3 and the side-chain oxygen of Thr217 of kinetic analysis of the four mutants T219S, T219V, T219G, and penicillopepsin-JT1 ~219! in inhibitor complexes ~James et al., T219A is also described. A short review on penicillopepsins in- 1985!—and is probably present in enzyme-substrate complexes— cludes a brief summary of part of this work ~Hofmann, 1998!. may play a major role in the rate enhancing effect of P3 ~Hofmann et al., 1988!. This hydrogen bond is also present in substrate- analog complexes of endothiapepsin ~Foundling et al., 1987!, rhi- Results and discussion zopuspepsin ~Suguna et al., 1987!, human ~Fujinaga et al., 1995!, and porcine pepsins ~Abad-Zapatero et al., 1991; Chen et al., 1992!, Isolation and sequence of gene pepA chymosin ~Groves et al., 1998!, and candidapepsin, an aspartic proteinase from Candida albicans ~Cutfield et al., 1995!.Inca- The genomic library of P. janthinellum constructed in EMBL 3 thepsin D, a hydrogen bond between the oxygen of the analogous was screened with 32P-labeled ~0.5 1 1.6 kb! fragments that con- serine ~Ser235! and an inhibitor is also present but it forms with tain pepA, the gene encoding aspergillopepsin A ~Berka et al., the -C5O- of P4 instead of the -NH- of the same peptide bond 1990!, as described in Materials and methods. Aspergillopepsin A ~Baldwin et al., 1993!. There is a strong possibility that the hy- is 70% identical in amino acid sequence with penicillopepsin-JT1. drogen bond between the oxygen of the side chain of the fourth A total of 12 positive lambda clones were identified and purified residue C-terminal to the catalytic aspartic acid residue in the from 25,000 plaques. After restriction mapping, hybridization, sub- C-terminal domain of aspartic proteinases with the -NH- group of cloning, and sequencing, it was found that clone lambda-4,331 the P3 residue ~or the -C5O- of P4! of a substrate is a common contained the gene pepA, which codes for penicillopepsin-JT2 feature of all aspartic proteinases. In each of the 49 aspartic pro- ~Fig. 1!, whereas the other positive lambda clones analyzed con- teinases listed by Fusek and Vetvicka ~1995!, that position is oc- tained another gene pepB, which codes for penicillopepsin-JT3. cupied by an amino acid with a side chain carrying a hydrogen The nucleotide sequence, including 59 and 39 untranslated re- bond accepting oxygen, namely threonine, serine or asparagine, gions, of pepA for penicillopepsin-JT2 and the deduced amino acid the latter being present only in the aspartic proteinases from two sequence are shown in Figure 2. The coding region consists of species of Rhizomucor ~Fusek & Vetvicka, 1995!. Furthermore, a 1,299 nucleotides that include 2 short introns of 62 and 68 bp, serine or threonine is present at that position in all sequences that respectively. The location of the introns is based on the locations have been published since the compilation by Fusek and Vetvicka of introns 1 and 3 of the gene pepA from Aspergillus awamori and ~1995!. They include a number of enzymes from plants ~D’Hondt is confirmed by the alignment of the active enzyme part of the et al., 1997! and from animal parasites ~Becker et al., 1995; Harrop deduced amino acid sequence with the known amino acid se- et al., 1996!. In spite of the large sequence differences among the quences of penicillopepsin-JT1 ~Hsu et al., 1977; James & Sielecki, aspartic proteinases, such as the additional 100 residues that are 1983! and of aspergillopepsin A from A. awamori ~Berka et al., inserted in the plant enzymes ~Asakura et al., 1995!, there is only 1990!. The alignment of penicillopepsin-JT1 and penicillopepsin- one exception to the presence of a serine, threonine, or asparagine JT2 is shown in Figure 3. Their sequences are 71% identical. in that position in 111 sequences of aspartic proteinases of the Similarly, each of them is ;70% identical with aspergillopepsin A family A1 of Clan AA listed by Rawlings and Barrett ~1998!. The ~not shown!. Further support for the intron locations comes from exception is an allergen from the German cockroach ~Blattella the presence of the GTRNGT sequence, the 59 splice donor site, at germanica!, which also acts as an enzyme ~Arruda et al., 1995!. the start and the YAG sequence, the 39-splice acceptor site, at the The sequence around the catalytic aspartic acids in all other en- end of the introns.