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Evidence for the interaction of valine-10 in cystatin C with the S2 subsite of cathepsin B

Lindahl, Peter; Ripoll, Daniel; Abrahamson, Magnus LU ; Mort, John S and Storer, Andrew (1994) In Biochemistry 33(14). p.4384-4392
Abstract
The interactions between wild-type or mutant recombinant forms of human cystatin C and rat cathepsin B were characterized by measuring progress curves for substrate hydrolysis in the presence of inhibitor. The investigation was guided by the use of computer modeling and explores the possibility that amino acid residues in the N-terminal region of cystatin C interact with substrate-binding regions in the target enzyme. With cystatin C that has Val-10 replaced by an Arg residue (VallOArg cystatin C), the inhibition constant, Ki, increased 3 1-fold if the isosteric substitution Glu-245 to Gln was made in cathepsin

B. When the wild-type form of the inhibitor was used, the corresponding effect on Ki was less than 2-fold. In a similar... (More)
The interactions between wild-type or mutant recombinant forms of human cystatin C and rat cathepsin B were characterized by measuring progress curves for substrate hydrolysis in the presence of inhibitor. The investigation was guided by the use of computer modeling and explores the possibility that amino acid residues in the N-terminal region of cystatin C interact with substrate-binding regions in the target enzyme. With cystatin C that has Val-10 replaced by an Arg residue (VallOArg cystatin C), the inhibition constant, Ki, increased 3 1-fold if the isosteric substitution Glu-245 to Gln was made in cathepsin

B. When the wild-type form of the inhibitor was used, the corresponding effect on Ki was less than 2-fold. In a similar study, using cathepsin B in which the substitution to Gln is instead at Glu-17 1, no such difference in how Ki is affected was observed. Both Glu-245 and Glu-171 are located in the S2 subsite of cathepsin B. The observed effects on Ki indicate that the additional positive charge introduced in VallOArg cystatin C is interacting with the negative charge on Glu-245 in cathepsin B when these two proteins form a complex; the cystatin variant is thus binding in a substratelike manner with this region of the enzyme. Indirectly, these results suggest that when native cystatin C and cathepsin B form a complex, Val-10 in the inhibitor interacts with the S2 subsite of the enzyme. A Ki value of 0.13 nM was obtained for the interaction of

VallOArg cystatin C with papain. Compared to the dissociation equilibrium constant for wild-type cystatin

C and papain, this value represents a 12 000-fold decrease in affinity. The corresponding effect with cathepsin B was only 15-fold, which presumably reflects the importance of the P2 position in binding of the N-terminal region of cystatin C to different cysteine proteinases. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Biochemistry
volume
33
issue
14
pages
4384 - 4392
publisher
The American Chemical Society
external identifiers
  • scopus:0028345437
ISSN
0006-2960
DOI
10.1021/bi00180a036
language
English
LU publication?
yes
id
5a79ad6a-5a84-43cd-991e-047c6c74ebc9 (old id 1108607)
date added to LUP
2008-07-24 13:57:03
date last changed
2017-10-01 03:56:47
@article{5a79ad6a-5a84-43cd-991e-047c6c74ebc9,
  abstract     = {The interactions between wild-type or mutant recombinant forms of human cystatin C and rat cathepsin B were characterized by measuring progress curves for substrate hydrolysis in the presence of inhibitor. The investigation was guided by the use of computer modeling and explores the possibility that amino acid residues in the N-terminal region of cystatin C interact with substrate-binding regions in the target enzyme. With cystatin C that has Val-10 replaced by an Arg residue (VallOArg cystatin C), the inhibition constant, Ki, increased 3 1-fold if the isosteric substitution Glu-245 to Gln was made in cathepsin<br/><br>
B. When the wild-type form of the inhibitor was used, the corresponding effect on Ki was less than 2-fold. In a similar study, using cathepsin B in which the substitution to Gln is instead at Glu-17 1, no such difference in how Ki is affected was observed. Both Glu-245 and Glu-171 are located in the S2 subsite of cathepsin B. The observed effects on Ki indicate that the additional positive charge introduced in VallOArg cystatin C is interacting with the negative charge on Glu-245 in cathepsin B when these two proteins form a complex; the cystatin variant is thus binding in a substratelike manner with this region of the enzyme. Indirectly, these results suggest that when native cystatin C and cathepsin B form a complex, Val-10 in the inhibitor interacts with the S2 subsite of the enzyme. A Ki value of 0.13 nM was obtained for the interaction of<br/><br>
VallOArg cystatin C with papain. Compared to the dissociation equilibrium constant for wild-type cystatin<br/><br>
C and papain, this value represents a 12 000-fold decrease in affinity. The corresponding effect with cathepsin B was only 15-fold, which presumably reflects the importance of the P2 position in binding of the N-terminal region of cystatin C to different cysteine proteinases.},
  author       = {Lindahl, Peter and Ripoll, Daniel and Abrahamson, Magnus and Mort, John S and Storer, Andrew},
  issn         = {0006-2960},
  language     = {eng},
  number       = {14},
  pages        = {4384--4392},
  publisher    = {The American Chemical Society},
  series       = {Biochemistry},
  title        = {Evidence for the interaction of valine-10 in cystatin C with the S2 subsite of cathepsin B},
  url          = {http://dx.doi.org/10.1021/bi00180a036},
  volume       = {33},
  year         = {1994},
}