Interaction of chicken cystatin with inactivated papains

Björk, Ingemar; Ylinenjärvi, K

doi:10.1042/bj2600061

Cited by 42 publications

(30 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The effect of these substitutions on the affinity of the inhibitor for inactivated papains was appreciably different. Like wild-type cystatin C, all three variants bound with lower affinity to the inactivated papains than to the active enzyme, the affinity decreasing with increasing size of the inactivating group [4,16]. However, the largest part of the decrease in affinity for the inactivated enzymes, -104-fold for S-(methylthio)-papain and 500-fold for S-(carbamoylmethyl)-papain, was observed on replacement of the glycine residue with alanine, whereas substitution with glutamic acid or tryptophan only resulted in a further 2-5-fold reduction in affinity for both inactivated papains.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Probing the functional role of the N-terminal region of cystatins by equilibrium and kinetic studies of the binding of Gly-11 variants of recombinant human cystatin C to target proteinases

Björk

Brieditis

Abrahamson

1995

Biochemical Journal

Self Cite

View full text Add to dashboard Cite

The interaction between cystatin C variants, in which the evolutionarily conserved Gly-11 residue was substituted by Ala, Glu or Trp, and the cysteine proteinases, papain, ficin, actinidin and cathepsin B, was characterized. The substitutions reduced the affinity of binding in a manner consistent with the Gly residue of the wild-type inhibitor, allowing the N-terminal region to adopt a conformation that was optimal for interaction with target proteinases. Replacement of Gly-11 by Ala resulted in only a 5- to 100-fold reduction in binding affinity. Comparison with the affinities of wild-type cystatin C lacking the N-terminal region indicated that even this small structural change affects the conformation of this region sufficiently to largely abolish its interaction with the weakly binding proteinases, actinidin and cathepsin B. However, the substitution allows interactions of appreciable strength between the N-terminal region and the tightly binding enzymes, papain or ficin. Replacement of Gly-11 with the larger Glu and Trp residues substantially decreased the affinity of binding to all enzymes, from 10(3)- to 10(5)-fold. These substitutions further affect the conformation of the N-terminal region, so that interactions of this region with papain and ficin are also essentially eliminated. The decreased affinities of the three cystatin C variants for papain, ficin and actinidin were due exclusively to increased dissociation rate constants. In contrast, the decreased affinity between cathepsin B and the Ala-11 variant, the only one for which rate constants could be determined with this enzyme, was due almost entirely to a decreased association rate constant. This behaviour is analogous to that observed for forms of cystatin C lacking the N-terminal region and supports the conclusion that the mode of interaction of this region with target proteinases varies with the enzyme as a result of structural differences in the active-site region of the latter.

show abstract

Section: Discussionmentioning

confidence: 99%

“…The cathepsin B preparation was a gift from Dr. John S. Mort, Shriners Hospital, Montreal, Canada. The inactivated papain forms, S-(methylthio)-papain and S-(carbamoylmethyl)-papain, were obtained as in previous work [4,16]. Near-u.v.…”

Section: Methodsmentioning

confidence: 99%

Probing the functional role of the N-terminal region of cystatins by equilibrium and kinetic studies of the binding of Gly-11 variants of recombinant human cystatin C to target proteinases

Björk

Brieditis

Abrahamson

1995

Biochemical Journal

Self Cite

View full text Add to dashboard Cite

show abstract

“…In the complex [19,20] both cystatin loops interact with conserved primed subsites adjacent to the papain catalytic residues; the initially flexible [I 391 amino-terminal segment (the 'trunk', see Fig. 9C) loops over the catalytic Cys25 residue of papain, whose sulphur atom can be alkylated with B $2- 33. only minor effects on the association constant [140], and interacts via two more amino-terminal residues with putative subsites S2 and S3. In contrast to bound substrates (see [141]), this inhibitor trunk is removed from the catalytic residues in the complex and thus is not cleavable (see Figs 9 C and 12C).…”

Section: Cystatin -Cysteine-proteinase Interactionmentioning

confidence: 99%

Natural protein proteinase inhibitors and their interaction with proteinases

Bode

Huber

1992

European Journal of Biochemistry

1,012

624

View full text Add to dashboard Cite

The substrate-like 'canonical' inhibition by the 'small' serine proteinase inhibitors and the productlike inhibition by the carboxypeptidase inhibitor have provided the only atomic models of protein inhibitor -proteinase interactions for about 15 years. The recently published structures of cystatin/ stefin -papain complexes and of hirudin -thrombin complexes reveal novel non-substrate-like interactions, In addition, the structure of pro-carboxypeptidase showes a mode of inactivation which bears resemblance to proteinase/protein inhibitor systems. Considerable progress in understanding the transition between native and cleaved states of the serpins has also been made by several recent structural studies.Proteinase inhibitors are important tools of nature for regulating the proteolytic activity of their target proteinases, for blocking these in emergency cases, or for signaling receptor interactions or clearance. Endogenous inhibitors appear to be always proteins; small non-proteinaceous inhibitors which impair the proteolytic activity of host proteinases are produced in microorganisms.The number of proteinaceous proteinase inhibitors isolated and identified so far is extremely large. In a now classical review paper, Laskowski and Kato [2] introduced for the first time a rational nomenclature by grouping these diverse inhibitors into distinct protein families. In the meantime, this list of families has considerably expanded with the advent of many new inhibitor species, and is still growing.The majority of protein inhibitors known and characterized so far are directed towards serine proteinases. Within the last few years, a large number of protein inhibitors of cysteine proteinases have also been discovered and characterized [3,4]. In contrast, only a few protein inhibitors directed towards metallo-proteinases (TIMP and PCI, see [5,61) or aspartyl proteinases (see [7][8][9]) are known to date. The azmacroglobulin family presents an exception, as these proteins can inhibit all of these proteinases according to a 'molecular trap' mechanism by virtue of a promiscuous 'bait region' (see [IOI).Until recently, X-ray crystal structures of only a few serine proteinase inhibitors, one carboxypeptidase inhibitor, and some of their complexes with cognate proteinases were available. The X-ray crystal structures of protein inhibitors published up to 1985 have been reviewed by Read and James A new aspect is provided by inhibitor structures elucidated by two-dimensional NMR methods (see [22, 231 for reviews). These data are often complementary to X-ray data, but are restricted to isolated inhibitors of relatively small molecular mass; until now, no NMR structure of a protein inhibitor has been reported for whch there is no X-ray structure available.In this review, we shall attempt to illuminate the characteristic structural properties conferring inhibitory activity to proteins. Nature has used diverse approaches to achieve proteinase inhibition. This is particularly well illustrated by some more recently published structures. In...

show abstract

“…The legumain family members are believed to have a protein fold quite unlike that of papain, and to be much more closely related to the caspases and gingipain (19). Although the active site cysteine residue could seem to be a common factor, it is known not to be required for the interaction of papain with cystatins (20). The present investigation was undertaken to elucidate the mechanism of inhibition of mammalian legumain by cystatins.…”

mentioning

confidence: 99%

Inhibition of Mammalian Legumain by Some Cystatins Is Due to a Novel Second Reactive Site

Alvarez-Fernandez

Barrett

Gerhartz

et al. 1999

Journal of Biological Chemistry

257

219

View full text Add to dashboard Cite

We have investigated the inhibition of the recently identified family C13 cysteine peptidase, pig legumain, by human cystatin C. The cystatin was seen to inhibit enzyme activity by stoichiometric 1:1 binding in competition with substrate. The K i value for the interaction was 0.20 nM, i.e. cystatin C had an affinity for legumain similar to that for the papain-like family C1 cysteine peptidase, cathepsin B. However, cystatin C variants with alterations in the N-terminal region and the "second hairpin loop" that rendered the cystatin inactive against cathepsin B, still inhibited legumain with K i values 0.2-0.3 nM. Complexes between cystatin C and papain inhibited legumain activity against benzoyl-AsnNHPhNO 2 as efficiently as did cystatin C alone. Conversely, cystatin C inhibited papain activity against benzoyl-Arg-NHPhNO 2 whether or not the cystatin had been incubated with legumain, strongly indicating that the cystatin inhibited the two enzymes with non-overlapping sites. A ternary complex between legumain, cystatin C, and papain was demonstrated by gel filtration supported by immunoblotting. Screening of a panel of cystatin superfamily members showed that type 1 inhibitors (cystatins A and B) and low M r kininogen (type 3) did not inhibit pig legumain. Of human type 2 cystatins, cystatin D was non-inhibitory, whereas cystatin E/M and cystatin F displayed strong (K i 0.0016 nM) and relatively weak (K i 10 nM) affinity for legumain, respectively. Sequence alignments and molecular modeling led to the suggestion that a loop located on the opposite side to the papain-binding surface, between the ␣-helix and the first strand of the main ␤-pleated sheet of the cystatin structure, could be involved in legumain binding. This was corroborated by analysis of a cystatin C variant with substitution of the Asn 39 residue in this loop (N39K-cystatin C); this variant showed a slight reduction in affinity for cathepsin B (K i 1.5 nM) but > >5,000-fold lower affinity for legumain (K i > >1,000 nM) than wild-type cystatin C.

show abstract

Interaction of chicken cystatin with inactivated papains

Cited by 42 publications

References 18 publications

Probing the functional role of the N-terminal region of cystatins by equilibrium and kinetic studies of the binding of Gly-11 variants of recombinant human cystatin C to target proteinases

Probing the functional role of the N-terminal region of cystatins by equilibrium and kinetic studies of the binding of Gly-11 variants of recombinant human cystatin C to target proteinases

Natural protein proteinase inhibitors and their interaction with proteinases

Inhibition of Mammalian Legumain by Some Cystatins Is Due to a Novel Second Reactive Site

Contact Info

Product

Resources

About