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. 1995 Apr;4(4):689–702. doi: 10.1002/pro.5560040409

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.

W T Lowther 1, P Majer 1, B M Dunn 1
PMCID: PMC2143106  PMID: 7613467

Abstract

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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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Azuma T., Liu W. G., Vander Laan D. J., Bowcock A. M., Taggart R. T. Human gastric cathepsin E gene. Multiple transcripts result from alternative polyadenylation of the primary transcripts of a single gene locus at 1q31-q32. J Biol Chem. 1992 Jan 25;267(3):1609–1614. [PubMed] [Google Scholar]
  2. Balbaa M., Cunningham A., Hofmann T. Secondary substrate binding in aspartic proteinases: contributions of subsites S3 and S'2 to kcat. Arch Biochem Biophys. 1993 Nov 1;306(2):297–303. doi: 10.1006/abbi.1993.1515. [DOI] [PubMed] [Google Scholar]
  3. Blundell T. L., Cooper J., Foundling S. I., Jones D. M., Atrash B., Szelke M. On the rational design of renin inhibitors: X-ray studies of aspartic proteinases complexed with transition-state analogues. Biochemistry. 1987 Sep 8;26(18):5585–5590. doi: 10.1021/bi00392a001. [DOI] [PubMed] [Google Scholar]
  4. Carter P. J., Winter G., Wilkinson A. J., Fersht A. R. The use of double mutants to detect structural changes in the active site of the tyrosyl-tRNA synthetase (Bacillus stearothermophilus). Cell. 1984 Oct;38(3):835–840. doi: 10.1016/0092-8674(84)90278-2. [DOI] [PubMed] [Google Scholar]
  5. Chen K. C., Tao N., Tang J. Primary structure of porcine pepsin. I. Purification and placement of cyanogen bromide fragments and the amino acid sequence of fragment CB5. J Biol Chem. 1975 Jul 10;250(13):5068–5075. [PubMed] [Google Scholar]
  6. Chen Z., Koelsch G., Han H. P., Wang X. J., Lin X. L., Hartsuck J. A., Tang J. Recombinant rhizopuspepsinogen. Expression, purification, and activation properties of recombinant rhizopuspepsinogens. J Biol Chem. 1991 Jun 25;266(18):11718–11725. [PubMed] [Google Scholar]
  7. Cooper J., Quail W., Frazao C., Foundling S. I., Blundell T. L., Humblet C., Lunney E. A., Lowther W. T., Dunn B. M. X-ray crystallographic analysis of inhibition of endothiapepsin by cyclohexyl renin inhibitors. Biochemistry. 1992 Sep 8;31(35):8142–8150. doi: 10.1021/bi00150a005. [DOI] [PubMed] [Google Scholar]
  8. Davies D. R. The structure and function of the aspartic proteinases. Annu Rev Biophys Biophys Chem. 1990;19:189–215. doi: 10.1146/annurev.bb.19.060190.001201. [DOI] [PubMed] [Google Scholar]
  9. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Faust P. L., Kornfeld S., Chirgwin J. M. Cloning and sequence analysis of cDNA for human cathepsin D. Proc Natl Acad Sci U S A. 1985 Aug;82(15):4910–4914. doi: 10.1073/pnas.82.15.4910. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fusek M., Smith E. A., Monod M., Dunn B. M., Foundling S. I. Extracellular aspartic proteinases from Candida albicans, Candida tropicalis, and Candida parapsilosis yeasts differ substantially in their specificities. Biochemistry. 1994 Aug 16;33(32):9791–9799. doi: 10.1021/bi00198a051. [DOI] [PubMed] [Google Scholar]
  12. Graham J. E., Sodek J., Hofmann T. Rhizopus acid proteinases (rhizopus-pepsins): properties and homology with other acid proteinases. Can J Biochem. 1973 Jun;51(6):789–796. doi: 10.1139/o73-098. [DOI] [PubMed] [Google Scholar]
  13. Gráf L., Jancsó A., Szilágyi L., Hegyi G., Pintér K., Náray-Szabó G., Hepp J., Medzihradszky K., Rutter W. J. Electrostatic complementarity within the substrate-binding pocket of trypsin. Proc Natl Acad Sci U S A. 1988 Jul;85(14):4961–4965. doi: 10.1073/pnas.85.14.4961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hobart P. M., Fogliano M., O'Connor B. A., Schaefer I. M., Chirgwin J. M. Human renin gene: structure and sequence analysis. Proc Natl Acad Sci U S A. 1984 Aug;81(16):5026–5030. doi: 10.1073/pnas.81.16.5026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hofmann T., Hodges R. S., James M. N. Effect of pH on the activities of penicillopepsin and Rhizopus pepsin and a proposal for the productive substrate binding mode in penicillopepsin. Biochemistry. 1984 Feb 14;23(4):635–643. doi: 10.1021/bi00299a008. [DOI] [PubMed] [Google Scholar]
  16. Hube B., Turver C. J., Odds F. C., Eiffert H., Boulnois G. J., Köchel H., Rüchel R. Sequence of the Candida albicans gene encoding the secretory aspartate proteinase. J Med Vet Mycol. 1991;29(2):129–132. [PubMed] [Google Scholar]
  17. Hyland L. J., Tomaszek T. A., Jr, Roberts G. D., Carr S. A., Magaard V. W., Bryan H. L., Fakhoury S. A., Moore M. L., Minnich M. D., Culp J. S. Human immunodeficiency virus-1 protease. 1. Initial velocity studies and kinetic characterization of reaction intermediates by 18O isotope exchange. Biochemistry. 1991 Aug 27;30(34):8441–8453. doi: 10.1021/bi00098a023. [DOI] [PubMed] [Google Scholar]
  18. Jackson S. E., Moracci M., elMasry N., Johnson C. M., Fersht A. R. Effect of cavity-creating mutations in the hydrophobic core of chymotrypsin inhibitor 2. Biochemistry. 1993 Oct 26;32(42):11259–11269. doi: 10.1021/bi00093a001. [DOI] [PubMed] [Google Scholar]
  19. Lin X., Tang J., Koelsch G., Monod M., Foundling S. Recombinant canditropsin, an extracellular aspartic protease from yeast Candida tropicalis. Escherichia coli expression, purification, zymogen activation, and enzymic properties. J Biol Chem. 1993 Sep 25;268(27):20143–20147. [PubMed] [Google Scholar]
  20. Loh Y. P., Parish D. C., Tuteja R. Purification and characterization of a paired basic residue-specific pro-opiomelanocortin converting enzyme from bovine pituitary intermediate lobe secretory vesicles. J Biol Chem. 1985 Jun 25;260(12):7194–7205. [PubMed] [Google Scholar]
  21. Lowther W. T., Chen Z., Lin X. L., Tang J., Dunn B. M. Substrate specificity study of recombinant Rhizopus chinensis aspartic proteinase. Adv Exp Med Biol. 1991;306:275–279. doi: 10.1007/978-1-4684-6012-4_33. [DOI] [PubMed] [Google Scholar]
  22. Lunney E. A., Hamilton H. W., Hodges J. C., Kaltenbronn J. S., Repine J. T., Badasso M., Cooper J. B., Dealwis C., Wallace B. A., Lowther W. T. Analyses of ligand binding in five endothiapepsin crystal complexes and their use in the design and evaluation of novel renin inhibitors. J Med Chem. 1993 Nov 26;36(24):3809–3820. doi: 10.1021/jm00076a008. [DOI] [PubMed] [Google Scholar]
  23. Matthews B. W. Structural and genetic analysis of protein stability. Annu Rev Biochem. 1993;62:139–160. doi: 10.1146/annurev.bi.62.070193.001035. [DOI] [PubMed] [Google Scholar]
  24. Morihara K., Oka T. Comparative specificity of microbial acid proteinases for synthetic peptides. 3. Relationship with their trypsinogen activating ability. Arch Biochem Biophys. 1973 Aug;157(2):561–572. doi: 10.1016/0003-9861(73)90675-9. [DOI] [PubMed] [Google Scholar]
  25. Morrison C. J., Hurst S. F., Bragg S. L., Kuykendall R. J., Diaz H., McLaughlin D. W., Reiss E. Purification and characterization of the extracellular aspartyl proteinase of Candida albicans: removal of extraneous proteins and cell wall mannoprotein and evidence for lack of glycosylation. J Gen Microbiol. 1993 Jun;139(Pt 6):1177–1186. doi: 10.1099/00221287-139-6-1177. [DOI] [PubMed] [Google Scholar]
  26. Pace C. N. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 1986;131:266–280. doi: 10.1016/0076-6879(86)31045-0. [DOI] [PubMed] [Google Scholar]
  27. Parris K. D., Hoover D. J., Damon D. B., Davies D. R. Synthesis and crystallographic analysis of two rhizopuspepsin inhibitor complexes. Biochemistry. 1992 Sep 8;31(35):8125–8141. doi: 10.1021/bi00150a004. [DOI] [PubMed] [Google Scholar]
  28. Paya C. V. Fungal infections in solid-organ transplantation. Clin Infect Dis. 1993 May;16(5):677–688. doi: 10.1093/clind/16.5.677. [DOI] [PubMed] [Google Scholar]
  29. Pohl J., Dunn B. M. Secondary enzyme-substrate interactions: kinetic evidence for ionic interactions between substrate side chains and the pepsin active site. Biochemistry. 1988 Jun 28;27(13):4827–4834. doi: 10.1021/bi00413a037. [DOI] [PubMed] [Google Scholar]
  30. Rao C. M., Scarborough P. E., Kay J., Batley B., Rapundalo S., Klutchko S., Taylor M. D., Lunney E. A., Humblet C. C., Dunn B. M. Specificity in the binding of inhibitors to the active site of human/primate aspartic proteinases: analysis of P2-P1-P1'-P2' variation. J Med Chem. 1993 Sep 3;36(18):2614–2620. doi: 10.1021/jm00070a004. [DOI] [PubMed] [Google Scholar]
  31. Salituro F. G., Agarwal N., Hofmann T., Rich D. H. Inhibition of aspartic proteinases by peptides containing lysine and ornithine side-chain analogues of statine. J Med Chem. 1987 Feb;30(2):286–295. doi: 10.1021/jm00385a010. [DOI] [PubMed] [Google Scholar]
  32. Samaranayake L. P., Holmstrup P. Oral candidiasis and human immunodeficiency virus infection. J Oral Pathol Med. 1989 Dec;18(10):554–564. doi: 10.1111/j.1600-0714.1989.tb01552.x. [DOI] [PubMed] [Google Scholar]
  33. Sawyer T. K., Staples D. J., Liu L., Tomasselli A. G., Hui J. O., O'Connell K., Schostarez H., Hester J. B., Moon J., Howe W. J. HIV protease (HIV PR) inhibitor structure-activity-selectivity, and active site molecular modeling of high affinity Leu [CH(OH)CH2]Val modified viral and nonviral substrate analogs. Int J Pept Protein Res. 1992 Sep-Oct;40(3-4):274–281. doi: 10.1111/j.1399-3011.1992.tb00302.x. [DOI] [PubMed] [Google Scholar]
  34. Scarborough P. E., Dunn B. M. Redesign of the substrate specificity of human cathepsin D: the dominant role of position 287 in the S2 subsite. Protein Eng. 1994 Apr;7(4):495–502. doi: 10.1093/protein/7.4.495. [DOI] [PubMed] [Google Scholar]
  35. Scarborough P. E., Guruprasad K., Topham C., Richo G. R., Conner G. E., Blundell T. L., Dunn B. M. Exploration of subsite binding specificity of human cathepsin D through kinetics and rule-based molecular modeling. Protein Sci. 1993 Feb;2(2):264–276. doi: 10.1002/pro.5560020215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  37. Shortle D. Mutational studies of protein structures and their stabilities. Q Rev Biophys. 1992 May;25(2):205–250. doi: 10.1017/s0033583500004674. [DOI] [PubMed] [Google Scholar]
  38. Sogawa K., Fujii-Kuriyama Y., Mizukami Y., Ichihara Y., Takahashi K. Primary structure of human pepsinogen gene. J Biol Chem. 1983 Apr 25;258(8):5306–5311. [PubMed] [Google Scholar]
  39. Sugrue R., Marston F. A., Lowe P. A., Freedman R. B. Denaturation studies on natural and recombinant bovine prochymosin (prorennin). Biochem J. 1990 Oct 15;271(2):541–547. doi: 10.1042/bj2710541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Suguna K., Padlan E. A., Bott R., Boger J., Parris K. D., Davies D. R. Structures of complexes of rhizopuspepsin with pepstatin and other statine-containing inhibitors. Proteins. 1992 Jul;13(3):195–205. doi: 10.1002/prot.340130303. [DOI] [PubMed] [Google Scholar]
  41. Suguna K., Padlan E. A., Smith C. W., Carlson W. D., Davies D. R. Binding of a reduced peptide inhibitor to the aspartic proteinase from Rhizopus chinensis: implications for a mechanism of action. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7009–7013. doi: 10.1073/pnas.84.20.7009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Suzuki J., Sasaki K., Sasao Y., Hamu A., Kawasaki H., Nishiyama M., Horinouchi S., Beppu T. Alteration of catalytic properties of chymosin by site-directed mutagenesis. Protein Eng. 1989 May;2(7):563–569. doi: 10.1093/protein/2.7.563. [DOI] [PubMed] [Google Scholar]
  43. Togni G., Sanglard D., Falchetto R., Monod M. Isolation and nucleotide sequence of the extracellular acid protease gene (ACP) from the yeast Candida tropicalis. FEBS Lett. 1991 Jul 29;286(1-2):181–185. doi: 10.1016/0014-5793(91)80969-a. [DOI] [PubMed] [Google Scholar]
  44. Wells J. A. Additivity of mutational effects in proteins. Biochemistry. 1990 Sep 18;29(37):8509–8517. doi: 10.1021/bi00489a001. [DOI] [PubMed] [Google Scholar]
  45. de Viragh P. A., Sanglard D., Togni G., Falchetto R., Monod M. Cloning and sequencing of two Candida parapsilosis genes encoding acid proteases. J Gen Microbiol. 1993 Feb;139(2):335–342. doi: 10.1099/00221287-139-2-335. [DOI] [PubMed] [Google Scholar]

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