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Ben M Dunn - One of the best experts on this subject based on the ideXlab platform.
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Engineering the substrate specificity of Rhizopuspepsin: the role of Asp 77 of fungal aspartic proteinases in facilitating the cleavage of oligopeptide substrates with lysine in P1.
Protein science : a publication of the Protein Society, 2008Co-Authors: W. Todd Lowther, Ben M Dunn, Pavel MajerAbstract:Rhizopuspepsin and other fungal aspartic proteinases are distinct from the mammalian enzymes in that they are able to cleave substrates with lysine in the P1 position. Sequence and structural comparisons suggest that two aspartic acid residues, Asp 30 and Asp 77 (pig pepsin numbering), may be responsible for generating this unique specificity. Asp 30 and Asp 77 were changed to the corresponding residues in porcine pepsin, Ile 30 and Thr 77, to create single and double mutants. The zymogen forms of the wild-type and mutant enzymes were overexpressed in Escherichia coli as inclusion bodies. Following solubilization, denaturation, refolding, activation, and purification to homogeneity, structural and kinetic comparisons were made. The mutant enzymes exhibited a high degree of structural similarity to the wild-type recombinant protein and a native isozyme. The catalytic activities of the recombinant proteins were analyzed with chromogenic substrates containing lysine in the P1, P2, or P3 positions. Mutation of Asp 77 resulted in a loss of 7 kcal mol-1 of transition-state stabilization energy in the hydrolysis of the substrate containing lysine in P1. An inhibitor containing the positively charged P1-lysine side chain inhibited only the enzymes containing Asp 77. Inhibition of the Asp 77 mutants of Rhizopuspepsin and several mammalian enzymes was restored upon acetylation of the lysine side chain. These results suggest that an exploitation of the specific electrostatic interaction of Asp 77 in the active site of fungal enzymes may lead to the design of compounds that preferentially inhibit a variety of related Candida proteinases in immunocompromised patients.
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Comparison of The Specificity of the Aspartic Proteinases Towards Internally Consistent Sets of Oligopeptide Substrates
Advances in experimental medicine and biology, 1998Co-Authors: Ben M Dunn, W. Todd Lowther, Kohei Oda, John Kay, Chetana Rao-naik, Brian M. Beyer, Paula E. Scarborough, Marina BukhtiyarovaAbstract:At the 1993 conference in Gifu, the concept of direct comparison of catalytic specificity between enzymes of the aspartic proteinase class was introduced.1 To achieve this, sets of oligopeptide substrates were used for precise kinetic studies. The parent peptide was: Lys—Pro—Ala—Lys—Phe*Nph—Arg—Leu, with cleavage occurring between the Phe and Nph (p-nitrophenylalanine) residues in all cases. In that report, we presented the set of peptides created by varying the P5, P4, P3, P2, P2′ and P3′ residues of the parent sequence. We substituted Ala, Ser, Asp, Leu, and Arg in each position, thereby exploring a range of potential interactions. In our previous discussion, the critical role of the residues in the P3 and P2 positions of the substrate was emphasized. Three characteristic behaviors were noted for the enzymes; pig pepsin, human cathepsin D, and Rhizopuspepsin. Pepsin showed a broad specificity, but could not tolerate an Arg residue in P3 at low pH; cathepsin D demonstrated a much tighter specificity, being unable to cleave any peptide with the Lys residue in P2, but showing strong activity versus peptides with Leu or Ala in that position. Rhizopuspepsin possessed the broadest specificity of all, with rapid cleavage of all ten substrates in the set compared. In all cases, the highest activity measured for each enzyme, against the best substrate for that enzyme, was comparable: in the range of 0.8–2.4 × 106 M-1 sec-1 for kcat/Km.
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Site-directed mutagenesis of Rhizopuspepsin: an analysis of unique specificity.
Advances in experimental medicine and biology, 1995Co-Authors: W. Todd Lowther, Ben M DunnAbstract:Rhizopuspepsin is an aspartic proteinase from the fungus Rhizopus chinensis. Interest in Rhizopuspepsin as a model system for studying active site interactions stems principally from the need to understand interactions which may be exploited in rational drug design. Targets for these therapeutics are renin, which plays a role in hypertension, the HIV proteinase, essential to the HIV life cycle, and a variety of related, secreted Candida yeast aspartic proteinases[1, 2].
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Substrate Specificity Study of Recombinant Rhizopus Chinensis Aspartic Proteinase
Advances in Experimental Medicine and Biology, 1991Co-Authors: W. Todd Lowther, Jordan Tang, Zhong Chen, Ben M DunnAbstract:Rhizopuspepsin, a model aspartic proteinase from the fungus Rhizopus chinensis, has recently been cloned and expressed by Chen et al. (1991). High resolution crystallographic analysis of Rhizopuspepsin and complexes with active site ligands has been reported by Davies’ group (Parris et al., this volume). Our initial characterization of the substrate specificity of the active site is described in this report. This study will enable future comparisons between kinetic and crystallographic data from other aspartic proteinases as well as for use in planning and analyzing site-directed mutagenesis studies.
W. Todd Lowther - One of the best experts on this subject based on the ideXlab platform.
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Engineering the substrate specificity of Rhizopuspepsin: the role of Asp 77 of fungal aspartic proteinases in facilitating the cleavage of oligopeptide substrates with lysine in P1.
Protein science : a publication of the Protein Society, 2008Co-Authors: W. Todd Lowther, Ben M Dunn, Pavel MajerAbstract:Rhizopuspepsin and other fungal aspartic proteinases are distinct from the mammalian enzymes in that they are able to cleave substrates with lysine in the P1 position. Sequence and structural comparisons suggest that two aspartic acid residues, Asp 30 and Asp 77 (pig pepsin numbering), may be responsible for generating this unique specificity. Asp 30 and Asp 77 were changed to the corresponding residues in porcine pepsin, Ile 30 and Thr 77, to create single and double mutants. The zymogen forms of the wild-type and mutant enzymes were overexpressed in Escherichia coli as inclusion bodies. Following solubilization, denaturation, refolding, activation, and purification to homogeneity, structural and kinetic comparisons were made. The mutant enzymes exhibited a high degree of structural similarity to the wild-type recombinant protein and a native isozyme. The catalytic activities of the recombinant proteins were analyzed with chromogenic substrates containing lysine in the P1, P2, or P3 positions. Mutation of Asp 77 resulted in a loss of 7 kcal mol-1 of transition-state stabilization energy in the hydrolysis of the substrate containing lysine in P1. An inhibitor containing the positively charged P1-lysine side chain inhibited only the enzymes containing Asp 77. Inhibition of the Asp 77 mutants of Rhizopuspepsin and several mammalian enzymes was restored upon acetylation of the lysine side chain. These results suggest that an exploitation of the specific electrostatic interaction of Asp 77 in the active site of fungal enzymes may lead to the design of compounds that preferentially inhibit a variety of related Candida proteinases in immunocompromised patients.
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Comparison of The Specificity of the Aspartic Proteinases Towards Internally Consistent Sets of Oligopeptide Substrates
Advances in experimental medicine and biology, 1998Co-Authors: Ben M Dunn, W. Todd Lowther, Kohei Oda, John Kay, Chetana Rao-naik, Brian M. Beyer, Paula E. Scarborough, Marina BukhtiyarovaAbstract:At the 1993 conference in Gifu, the concept of direct comparison of catalytic specificity between enzymes of the aspartic proteinase class was introduced.1 To achieve this, sets of oligopeptide substrates were used for precise kinetic studies. The parent peptide was: Lys—Pro—Ala—Lys—Phe*Nph—Arg—Leu, with cleavage occurring between the Phe and Nph (p-nitrophenylalanine) residues in all cases. In that report, we presented the set of peptides created by varying the P5, P4, P3, P2, P2′ and P3′ residues of the parent sequence. We substituted Ala, Ser, Asp, Leu, and Arg in each position, thereby exploring a range of potential interactions. In our previous discussion, the critical role of the residues in the P3 and P2 positions of the substrate was emphasized. Three characteristic behaviors were noted for the enzymes; pig pepsin, human cathepsin D, and Rhizopuspepsin. Pepsin showed a broad specificity, but could not tolerate an Arg residue in P3 at low pH; cathepsin D demonstrated a much tighter specificity, being unable to cleave any peptide with the Lys residue in P2, but showing strong activity versus peptides with Leu or Ala in that position. Rhizopuspepsin possessed the broadest specificity of all, with rapid cleavage of all ten substrates in the set compared. In all cases, the highest activity measured for each enzyme, against the best substrate for that enzyme, was comparable: in the range of 0.8–2.4 × 106 M-1 sec-1 for kcat/Km.
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Site-directed mutagenesis of Rhizopuspepsin: an analysis of unique specificity.
Advances in experimental medicine and biology, 1995Co-Authors: W. Todd Lowther, Ben M DunnAbstract:Rhizopuspepsin is an aspartic proteinase from the fungus Rhizopus chinensis. Interest in Rhizopuspepsin as a model system for studying active site interactions stems principally from the need to understand interactions which may be exploited in rational drug design. Targets for these therapeutics are renin, which plays a role in hypertension, the HIV proteinase, essential to the HIV life cycle, and a variety of related, secreted Candida yeast aspartic proteinases[1, 2].
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Substrate Specificity Study of Recombinant Rhizopus Chinensis Aspartic Proteinase
Advances in Experimental Medicine and Biology, 1991Co-Authors: W. Todd Lowther, Jordan Tang, Zhong Chen, Ben M DunnAbstract:Rhizopuspepsin, a model aspartic proteinase from the fungus Rhizopus chinensis, has recently been cloned and expressed by Chen et al. (1991). High resolution crystallographic analysis of Rhizopuspepsin and complexes with active site ligands has been reported by Davies’ group (Parris et al., this volume). Our initial characterization of the substrate specificity of the active site is described in this report. This study will enable future comparisons between kinetic and crystallographic data from other aspartic proteinases as well as for use in planning and analyzing site-directed mutagenesis studies.
Wen-hwei Hsu - One of the best experts on this subject based on the ideXlab platform.
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Purification and characterization of a new Rhizopuspepsin from Rhizopus oryzae NBRC 4749.
Journal of agricultural and food chemistry, 2009Co-Authors: Chun-chang Chen, Yen-ching Cho, Chien-chen Lai, Wen-hwei HsuAbstract:A secretory aspartic protease (also termed as Rhizopuspepsin) was purified from Rhizopus oryzae NBRC 4749 by ion exchange chromatography with a yield of 45%. The enzyme was a nonglycoprotein with a molecular mass of 37 kDa as determined by SDS−PAGE analysis. N-terminal sequence and LC-MS/MS analyses revealed that this Rhizopuspepsin corresponded to the hypothetical protein RO3G_12822.1 in the R. oryzae genome database. Comparison of genomic and cDNA genes demonstrated that the Rhizopuspepsin contained two introns, whereas only one intron was reported in other Rhizopuspepsin genes. Phylogenetic analysis also indicated that this Rhizopuspepsin was distinct from other Rhizopuspepsins. The temperature and pH optima for the purified Rhizopuspepsin were 50 °C and pH 3.0, respectively, and a half-life of about 3.5 h was observed at 40 °C. The enzyme preferentially cleaved the peptides with hydrophobic and basic amino acids in the P1 site but had no activity for the Glu, Pro, Trp, and aliphatic amino acids contai...
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purification and characterization of a new Rhizopuspepsin from rhizopus oryzae nbrc 4749
Journal of Agricultural and Food Chemistry, 2009Co-Authors: Chun-chang Chen, Yen-ching Cho, Chien-chen Lai, Wen-hwei HsuAbstract:A secretory aspartic protease (also termed as Rhizopuspepsin) was purified from Rhizopus oryzae NBRC 4749 by ion exchange chromatography with a yield of 45%. The enzyme was a nonglycoprotein with a molecular mass of 37 kDa as determined by SDS-PAGE analysis. N-terminal sequence and LC-MS/MS analyses revealed that this Rhizopuspepsin corresponded to the hypothetical protein RO3G_12822.1 in the R. oryzae genome database. Comparison of genomic and cDNA genes demonstrated that the Rhizopuspepsin contained two introns, whereas only one intron was reported in other Rhizopuspepsin genes. Phylogenetic analysis also indicated that this Rhizopuspepsin was distinct from other Rhizopuspepsins. The temperature and pH optima for the purified Rhizopuspepsin were 50 degrees C and pH 3.0, respectively, and a half-life of about 3.5 h was observed at 40 degrees C. The enzyme preferentially cleaved the peptides with hydrophobic and basic amino acids in the P1 site but had no activity for the Glu, Pro, Trp, and aliphatic amino acids containing the beta-branch side chain.
David R Davies - One of the best experts on this subject based on the ideXlab platform.
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Synthesis and crystallographic analysis of two Rhizopuspepsin inhibitor complexes.
Biochemistry, 1992Co-Authors: Kevin D. Parris, Hoover Dennis Jay, David B. Damon, David R DaviesAbstract:The crystal structures of Rhizopuspepsin complexed with two oligopeptide inhibitors have been determined. CP-69,799, an azahomostatine dipeptide isostere, had previously been associated with a displacement of the C-terminal subdomain of endothiapepsin [Sali, A., Veerapandian, B., Cooper, J. B., Foundling, S. I., Hoover, D. J., & Blundell, T. L. (1989) EMBO J. 8, 2179-2188]. Here, we report the measurement of two data sets, one from crystals soaked in the inhibitor and the other from protein crystallized in the presence of excess inhibitor. In neither case is there any significant movement of the C-terminal subdomain of the Rhizopuspepsin. The data suggest that the energy associated with any conformational change is small and is overcome by the crystal packing forces. The second inhibitor, a hydrated difluorostatone, was examined in a search for transition-state analogs that could cast further light on the mechanism of action [Suguna, K., Padlan, E. A., Smith, C. W., Carlson, W. D., & Davies, D. R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7009-7013]. The gem-diol provides a set of contact distances with the enzyme that mimic the interactions with the tetrahedral intermediate of the substrate during catalysis. These data provide support for the suggestion that the polarization of the keto group of the peptide substrate is enhanced by a hydrogen bond from the OD1 of Asp 35 (Suguna et al., 1987).
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Structures of complexes of Rhizopuspepsin with pepstatin and other statine‐containing inhibitors
Proteins, 1991Co-Authors: Kaza Suguna, Eduardo A Padlan, Kevin D. Parris, Richard Bott, Joshua Boger, David R DaviesAbstract:The three-dimensional structures of the complexes of the aspartic proteinase from Rhizopus chinensis (Rhizopuspepsin, EC 3.4.23.6) with pepstatin and two pepstatin like peptide inhibitors of renin have been detrmined by X-ray diffraction methods and refined by restrained least-squares procedures. The inhibitors adopt an extended conformation and lie in the deep groove located between the two domains of the enzyme. Inhibitor binding is accompanied by a conformational change at the “flap,” a β-hairpin loop regions, that projects over the binding cleft andcloses down over the inhibitor, excluding water molecules from the vicinityof the scissile bond. The hydroxyl group of the central statyl residue of the inhibitors replaces the water molecule found between the two active aspartates, Asp-35 and Asp-218, in the native structure. The refined structures provide additional data to define the specific subsites of the enzyme and also show a system of hydrogen bonding to the inhibitor backbone similar to that observed for a reduced inhibitor. Published 1992 Wiley-Liss, Inc.
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structures of complexes of Rhizopuspepsin with pepstatin and other statine containing inhibitors
Proteins, 1991Co-Authors: Kaza Suguna, Eduardo A Padlan, David R Davies, Kevin D. Parris, Richard Bott, Joshua BogerAbstract:The three-dimensional structures of the complexes of the aspartic proteinase from Rhizopus chinensis (Rhizopuspepsin, EC 3.4.23.6) with pepstatin and two pepstatin like peptide inhibitors of renin have been detrmined by X-ray diffraction methods and refined by restrained least-squares procedures. The inhibitors adopt an extended conformation and lie in the deep groove located between the two domains of the enzyme. Inhibitor binding is accompanied by a conformational change at the “flap,” a β-hairpin loop regions, that projects over the binding cleft andcloses down over the inhibitor, excluding water molecules from the vicinityof the scissile bond. The hydroxyl group of the central statyl residue of the inhibitors replaces the water molecule found between the two active aspartates, Asp-35 and Asp-218, in the native structure. The refined structures provide additional data to define the specific subsites of the enzyme and also show a system of hydrogen bonding to the inhibitor backbone similar to that observed for a reduced inhibitor. Published 1992 Wiley-Liss, Inc.
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Crystal structures of Rhizopuspepsin/inhibitor complexes.
Advances in experimental medicine and biology, 1991Co-Authors: Kevin D. Parris, Dennis J. Hoover, David R DaviesAbstract:The crystal structures of the aspartic proteinases have been extensively studied over the past fifteen years (Hsu et al., 1977; Subramanian et al., 1977; reviewed by Davies, 1990). After the initial determinations of the native structures uncomplexed with inhibitors, a few complexes with pepstatin (Bott et al., 1982) and with a fragment of pepstatin (James et al., 1982) were reported. These studies showed that these inhibitors bound in the deep groove that separates the N- and C-terminal domains of the enzyme. Accompanying the binding of the inhibitor was a displacement of the “flap” region of the molecule, a hairpin loop that closes down on the inhibitor, the extent of the displacement depending on the initial location of the flap (James et al., 1982; Bott et al., 1982; Suguna et al., 1987 and Cooper et al., 1987). Since no other major conformational changes were observed, the crystals of these proteinases offered a convenient vehicle for examining a number of bound inhibitor conformations. Other factors facilitating the examination of these inhibitors were the availability of large numbers of renin inhibitors, and the fortunate ease of access to the combining site in several of the crystal forms, thus enabling the inhibitors to be soaked into the crystals.
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crystal structures of Rhizopuspepsin inhibitor complexes
Advances in Experimental Medicine and Biology, 1991Co-Authors: Kevin D. Parris, Dennis J. Hoover, David R DaviesAbstract:The crystal structures of the aspartic proteinases have been extensively studied over the past fifteen years (Hsu et al., 1977; Subramanian et al., 1977; reviewed by Davies, 1990). After the initial determinations of the native structures uncomplexed with inhibitors, a few complexes with pepstatin (Bott et al., 1982) and with a fragment of pepstatin (James et al., 1982) were reported. These studies showed that these inhibitors bound in the deep groove that separates the N- and C-terminal domains of the enzyme. Accompanying the binding of the inhibitor was a displacement of the “flap” region of the molecule, a hairpin loop that closes down on the inhibitor, the extent of the displacement depending on the initial location of the flap (James et al., 1982; Bott et al., 1982; Suguna et al., 1987 and Cooper et al., 1987). Since no other major conformational changes were observed, the crystals of these proteinases offered a convenient vehicle for examining a number of bound inhibitor conformations. Other factors facilitating the examination of these inhibitors were the availability of large numbers of renin inhibitors, and the fortunate ease of access to the combining site in several of the crystal forms, thus enabling the inhibitors to be soaked into the crystals.
Jordan Tang - One of the best experts on this subject based on the ideXlab platform.
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pH dependence of kinetic parameters of pepsin, Rhizopuspepsin, and their active-site hydrogen bond mutants.
The Journal of biological chemistry, 1992Co-Authors: Y Lin, Martin Fusek, Xinli Lin, J. A. Hartsuck, F. J. Kezdy, Jordan TangAbstract:The pH dependence of the kinetic parameters of pepsin, Rhizopuspepsin, and their active-site hydrogen bond mutants has been determined. These data have permitted the calculation of two active-site ionization constants in the free enzymes (pKe1 and pK32) and in the enzyme-substrate complexes (pKes1 and pKes2). The pKe1 of Rhizopuspepsin (2.8) is near that of a normal carboxyl group and near the pKe1 of human immunodeficiency virus type 1 (HIV-1) protease (3.32) (Ido, E., Han, H. P., Kezdy, F. J., and Tang, J. (1991) J. Biol. Chem. 266, 24359-24366). The pKe1 of pepsin (1.57) is thus abnormally low. The pKe2 of Rhizopuspepsin (4.44) is lower than that of pepsin (5.02) and HIV protease (6.80). The binding of substrate to Rhizopuspepsin causes the lowering of pKes1 to 1.8 and the elevating of pKes2 to above 6. The pK alpha shifts due to substrate binding are much less pronounced in pepsin. Thus, the two enzyme-substrate complexes have similar pK alpha values. For both pepsin and Rhizopuspepsin, the removal of hydrogen bonds to the active-site carboxyls by mutagenesis results in negligible changes in the four pK alpha values. The major alteration caused by these mutations is the decrease in kcat values, while there is little change in Km. These observations suggest that these hydrogen bonds to the active-site aspartyls contribute little to the pH-activity relationships of the aspartic proteases. The role of the active-site hydrogen bonds may well be to preserve the conformational rigidity of the catalytic apparatus.
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Substrate Specificity Study of Recombinant Rhizopus Chinensis Aspartic Proteinase
Advances in Experimental Medicine and Biology, 1991Co-Authors: W. Todd Lowther, Jordan Tang, Zhong Chen, Ben M DunnAbstract:Rhizopuspepsin, a model aspartic proteinase from the fungus Rhizopus chinensis, has recently been cloned and expressed by Chen et al. (1991). High resolution crystallographic analysis of Rhizopuspepsin and complexes with active site ligands has been reported by Davies’ group (Parris et al., this volume). Our initial characterization of the substrate specificity of the active site is described in this report. This study will enable future comparisons between kinetic and crystallographic data from other aspartic proteinases as well as for use in planning and analyzing site-directed mutagenesis studies.
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Recombinant Rhizopuspepsinogen. Expression, purification, and activation properties of recombinant Rhizopuspepsinogens.
The Journal of biological chemistry, 1991Co-Authors: Zhong Chen, Xinli Lin, J. A. Hartsuck, Gerald Koelsch, He-ping Han, Xin-juan Wang, Jordan TangAbstract:Abstract A cDNA clone, which contained the complete Rhizopuspepsin structure and the putative proregion, was placed in three different Escherichia coli expression vectors for the synthesis of Rhizopuspepsinogen (Rpg). Recombinant Rpgs which were expressed in the cytosol of E. coli as inclusion bodies (cRpg and tRpg) were not active. After solubilization in 6 M urea and refolding by rapid dilution, both of these Rpgs were purified to homogeneity. The third zymogen, pRpg, which was secreted to the periplasmic space of E. coli with an omp leader, was fully active and also was purified. The expression level of pRpg was higher (over 40 mg/liter culture) than that of cRpg (about 1.5 mg/liter culture). Amino-terminal sequence analysis of the zymogens revealed that cRpg and pRpg contain 40 and 51 residues of prosequence, respectively. tRpg, which was expressed under the control of T7 promoter, was synthesized at 500 mg/liter culture and was purified at 50 mg/liter culture. This zymogen contained, in addition to 51 residues of proregion, 16 residues inherited from the expression vector construction. All of these Rpgs spontaneously converted to Rhizopuspepsin in solutions of pH less than 5. Each of the conversions was associated with a change of molecular weight as monitored in sodium dodecyl sulfate-polyacrylamide electrophoresis. At least one intermediate of conversion was observed in the pH range of 2 to 3 for both the cRpg and pRpg zymogens. For pRpg and tRpg, kinetic data demonstrated that the Rpg to Rhizopuspepsin conversion was accomplished by a first order, unimolecular reaction at pH 2. The first order kinetic constants in this pH at 15 degrees C were 1.1 and 2.4 min-1 for pRpg and tRpg, respectively. The activation rate decreased as pH was raised above pH 2. At pH greater than 3.0, Rhizopuspepsin-catalyzed, second-order activation also takes place. Consequently, the recombinant Rpgs are activated by either of two cleavage mechanisms as is the case for pepsinogen. These results also support the hypothesis that Rpg is synthesized in Rhizopus chinensis as a zymogen. Rpg in the host fungus is probably activated by an acid environment of pH less than 5 in the secretory granules to become Rhizopuspepsin before secretion.