Logo of biochemjBJ Latest papers and much more!
Biochem J. 1994 Sep 1; 302(Pt 2): 411–416.
PMCID: PMC1137243

Local pH-dependent conformational changes leading to proteolytic susceptibility of cystatin C.


Cystatin C, a cysteine protease inhibitor, was subject to hydrolysis at two sites when complexed with papain and in the presence of excess papain. A pH-dependent cleavage at His-86 increases Asp-87 was observed, as well as a pH-independent one at Gly-4 increases Lys-5. His-86 increases Asp-87 hydrolysis increased with decreasing pH and was characterized kinetically. It could be described by a single ionization with pKa = 3.4 +/- 0.2 and (kcat./Km)max. = 1.4 (+/- 0.4) x 10(4) M-1.s-1 at I = 0.3 M. C.d. spectroscopy, also at I = 0.3 M, demonstrated a conformational change with pKa = 3.2 +/- 0.2, indicating that the pH-dependence of hydrolysis was due to a conformational change in cystatin C. At I = 0.15 M, the pKa of the conformational change observed by c.d. shifted to 4.1 +/- 0.1. This indicates that at physiological ionic strength of 0.15 M, a significant proportion of cystatin C complexed with protease would be in a proteolytically labile conformation over the pH range 4.5 to 5, which is encountered in lysosomes. This may constitute a mechanism for clearing inappropriately localized cystatins. A pH-dependent conformational variability in this region of the inhibitor could explain the differences in the X-ray crystallographic and n.m.r. structures of the homologous chicken cystatin. The ionic-strength dependence of ionization indicates a hydrophobic stabilization of the ionizable group. The lack of pH-dependence of hydrolysis at Gly-4 increases Lys-5, with kcat./Km = 220 +/- 41 M-1.s-1 in the pH range 3.89 to 7.96 was unexpected in light of the normal, bell-shaped pH-dependence of papain-catalysed hydrolyses. This may reflect a different rate-limiting step of cystatin C hydrolysis.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.1M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Turk V, Bode W. The cystatins: protein inhibitors of cysteine proteinases. FEBS Lett. 1991 Jul 22;285(2):213–219. [PubMed]
  • Lustigman S, Brotman B, Huima T, Prince AM, McKerrow JH. Molecular cloning and characterization of onchocystatin, a cysteine proteinase inhibitor of Onchocerca volvulus. J Biol Chem. 1992 Aug 25;267(24):17339–17346. [PubMed]
  • Irvine JW, Coombs GH, North MJ. Cystatin-like cysteine proteinase inhibitors of parasitic protozoa. FEMS Microbiol Lett. 1992 Sep 1;75(1):67–72. [PubMed]
  • Abrahamson M, Barrett AJ, Salvesen G, Grubb A. Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids. J Biol Chem. 1986 Aug 25;261(24):11282–11289. [PubMed]
  • Lindahl P, Abrahamson M, Björk I. Interaction of recombinant human cystatin C with the cysteine proteinases papain and actinidin. Biochem J. 1992 Jan 1;281(Pt 1):49–55. [PMC free article] [PubMed]
  • Björk I, Alriksson E, Ylinenjärvi K. Kinetics of binding of chicken cystatin to papain. Biochemistry. 1989 Feb 21;28(4):1568–1573. [PubMed]
  • Bode W, Engh R, Musil D, Thiele U, Huber R, Karshikov A, Brzin J, Kos J, Turk V. The 2.0 A X-ray crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine proteinases. EMBO J. 1988 Aug;7(8):2593–2599. [PMC free article] [PubMed]
  • Stubbs MT, Laber B, Bode W, Huber R, Jerala R, Lenarcic B, Turk V. The refined 2.4 A X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction. EMBO J. 1990 Jun;9(6):1939–1947. [PMC free article] [PubMed]
  • Auerswald EA, Genenger G, Mentele R, Lenzen S, Assfalg-Machleidt I, Mitschang L, Oschkinat H, Fritz H. Purification and characterization of a chicken egg white cystatin variant expressed in an Escherichia coli pIN-III-ompA system. Eur J Biochem. 1991 Aug 15;200(1):131–138. [PubMed]
  • Popović T, Brzin J, Ritonja A, Turk V. Different forms of human cystatin C. Biol Chem Hoppe Seyler. 1990 Jul;371(7):575–580. [PubMed]
  • Machleidt W, Thiele U, Laber B, Assfalg-Machleidt I, Esterl A, Wiegand G, Kos J, Turk V, Bode W. Mechanism of inhibition of papain by chicken egg white cystatin. Inhibition constants of N-terminally truncated forms and cyanogen bromide fragments of the inhibitor. FEBS Lett. 1989 Jan 30;243(2):234–238. [PubMed]
  • Abrahamson M, Mason RW, Hansson H, Buttle DJ, Grubb A, Ohlsson K. Human cystatin C. role of the N-terminal segment in the inhibition of human cysteine proteinases and in its inactivation by leucocyte elastase. Biochem J. 1991 Feb 1;273(Pt 3):621–626. [PMC free article] [PubMed]
  • Buttle DJ, Ritonja A, Dando PM, Abrahamson M, Shaw EN, Wikstrom P, Turk V, Barrett AJ. Interactions of papaya proteinase IV with inhibitors. FEBS Lett. 1990 Mar 12;262(1):58–60. [PubMed]
  • Lindahl P, Nycander M, Ylinenjärvi K, Pol E, Björk I. Characterization by rapid-kinetic and equilibrium methods of the interaction between N-terminally truncated forms of chicken cystatin and the cysteine proteinases papain and actinidin. Biochem J. 1992 Aug 15;286(Pt 1):165–171. [PMC free article] [PubMed]
  • Datta S, Firtel RA. Identification of the sequences controlling cyclic AMP regulation and cell-type-specific expression of a prestalk-specific gene in Dictyostelium discoideum. Mol Cell Biol. 1987 Jan;7(1):149–159. [PMC free article] [PubMed]
  • Koga H, Yamada H, Nishimura Y, Kato K, Imoto T. Comparative study on specificities of rat cathepsin L and papain: amino acid differences at substrate-binding sites are involved in their specificities. J Biochem. 1990 Dec;108(6):976–982. [PubMed]
  • Dalbøge H, Jensen EB, Tøttrup H, Grubb A, Abrahamson M, Olafsson I, Carlsen S. High-level expression of active human cystatin C in Escherichia coli. Gene. 1989 Jul 15;79(2):325–332. [PubMed]
  • Sluyterman LA, Wijdenes J. An agarose mercurial column for the separation of mercaptopapain and nonmercaptopapain. Biochim Biophys Acta. 1970 Mar 31;200(3):593–595. [PubMed]
  • Brocklehurst K, Carlsson J, Kierstan MP, Crook EM. Covalent chromatography. Preparation of fully active papain from dried papaya latex. Biochem J. 1973 Jul;133(3):573–584. [PMC free article] [PubMed]
  • Blumberg S, Schechter I, Berger A. The purification of papain by affinity chromatography. Eur J Biochem. 1970 Jul;15(1):97–102. [PubMed]
  • Feng R, Konishi Y. Analysis of antibodies and other large glycoproteins in the mass range of 150,000-200,000 Da by electrospray ionization mass spectrometry. Anal Chem. 1992 Sep 15;64(18):2090–2095. [PubMed]
  • Hewick RM, Hunkapiller MW, Hood LE, Dreyer WJ. A gas-liquid solid phase peptide and protein sequenator. J Biol Chem. 1981 Aug 10;256(15):7990–7997. [PubMed]
  • Cornish-Bowden A. The use of the direct linear plot for determining initial velocities. Biochem J. 1975 Aug;149(2):305–312. [PMC free article] [PubMed]
  • Selwyn MJ. A simple test for inactivation of an enzyme during assay. Biochim Biophys Acta. 1965 Jul 29;105(1):193–195. [PubMed]
  • Lindahl P, Alriksson E, Jörnvall H, Björk I. Interaction of the cysteine proteinase inhibitor chicken cystatin with papain. Biochemistry. 1988 Jul 12;27(14):5074–5082. [PubMed]
  • Johnson WC., Jr Protein secondary structure and circular dichroism: a practical guide. Proteins. 1990;7(3):205–214. [PubMed]
  • Desmazeaud MJ. Contribution à l'étude de la spécificité de la papaïne: hydrolyse du glucagon. Biochimie. 1972;54(9):1109–1114. [PubMed]
  • Johansen JT, Ottesen M. The proteolytic degradation of the B-chain of oxidized insulin by papain, chymopapain and papaya peptidase. C R Trav Lab Carlsberg. 1968;36(15):265–283. [PubMed]
  • Shaw E. Cysteinyl proteinases and their selective inactivation. Adv Enzymol Relat Areas Mol Biol. 1990;63:271–347. [PubMed]
  • Ménard R, Plouffe C, Khouri HE, Dupras R, Tessier DC, Vernet T, Thomas DY, Storer AC. Removal of an inter-domain hydrogen bond through site-directed mutagenesis: role of serine 176 in the mechanism of papain. Protein Eng. 1991 Feb;4(3):307–311. [PubMed]
  • Bode W, Brzin J, Turk V. Crystallization of chicken egg white cystatin, a low molecular weight protein inhibitor of cysteine proteinases, and preliminary X-ray diffraction data. J Mol Biol. 1985 Jan 20;181(2):331–332. [PubMed]
  • Björk I, Ylinenjärvi K. Different roles of the two disulfide bonds of the cysteine proteinase inhibitor, chicken cystatin, for the conformation of the active protein. Biochemistry. 1992 Sep 15;31(36):8597–8602. [PubMed]
  • Mellman I, Fuchs R, Helenius A. Acidification of the endocytic and exocytic pathways. Annu Rev Biochem. 1986;55:663–700. [PubMed]
  • Delshammar M, Lasson A, Ohlsson K. Proteases and protease inhibitor balance in peritonitis with different causes. Surgery. 1989 Sep;106(3):555–562. [PubMed]
  • von Figura K, Hasilik A. Lysosomal enzymes and their receptors. Annu Rev Biochem. 1986;55:167–193. [PubMed]
  • Dean RT, Barrett AJ. Lysosomes. Essays Biochem. 1976;12:1–40. [PubMed]
  • Oude Elferink RP, Van Doorn-Van Wakeren J, Hendriks T, Strijland A, Tager JM. Transport and processing of endocytosed lysosomal alpha-glucosidase in cultured human skin fibroblasts. Eur J Biochem. 1986 Jul 15;158(2):339–344. [PubMed]
  • Willingham MC, Pastan IH, Sahagian GG, Jourdian GW, Neufeld EF. Morphologic study of the internalization of a lysosomal enzyme by the mannose 6-phosphate receptor in cultured Chinese hamster ovary cells. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6967–6971. [PMC free article] [PubMed]
  • Prence EM, Dong JM, Sahagian GG. Modulation of the transport of a lysosomal enzyme by PDGF. J Cell Biol. 1990 Feb;110(2):319–326. [PMC free article] [PubMed]
  • Kominami E, Tsukahara T, Hara K, Katunuma N. Biosyntheses and processing of lysosomal cysteine proteinases in rat macrophages. FEBS Lett. 1988 Apr 11;231(1):225–228. [PubMed]
  • Tooze J, Hollinshead M, Hensel G, Kern HF, Hoflack B. Regulated secretion of mature cathepsin B from rat exocrine pancreatic cells. Eur J Cell Biol. 1991 Dec;56(2):187–200. [PubMed]
  • Mellor GW, Patel M, Thomas EW, Brocklehurst K. Clarification of the pH-dependent kinetic behaviour of papain by using reactivity probes and analysis of alkylation and catalysed acylation reactions in terms of multihydronic state models: implications for electrostatics calculations and interpretation of the consequences of site-specific mutations such as Asp-158-Asn and Asp-158-Glu. Biochem J. 1993 Aug 15;294(Pt 1):201–210. [PMC free article] [PubMed]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society


Save items

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Compound
    PubChem chemical compound records that cite the current articles. These references are taken from those provided on submitted PubChem chemical substance records. Multiple substance records may contribute to the PubChem compound record.
  • MedGen
    Related information in MedGen
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem chemical substance records that cite the current articles. These references are taken from those provided on submitted PubChem chemical substance records.

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...