Cathepsin B & Synthetic Substrates

[Simon M. Brocklehurst]

Louis Hom wrote:
> 
> So I've been looking at a viral protease that is homologous to the
> cathepsins, and has the substrate specificity of cathepsin B (i.e., it
> prefers an Arg 2 aa's N-terminal to the site of cleavage).  The researchers
> used Z-Arg-Arg-MCA and some other synthetic substrates to demonstrate this.
> What sort of puzzles me is why these experiments almost always use
> Z-Arg-X-MCA rather than Z-Lys-X-MCA.  After all, the crystal structure
> suggests a preference for the positive charge of Arg more than, say, its
> shape.  Any ideas?  Is there some esoteric quirk involving difficulty in
> synthesis?
> --

A couple of points...

  It is much easier to synthesise alpha-N-acyl-Arg derivatives than the
corresponding Lys ones-because of acylation of epsilon NH2 as well as alpha
NH2.

  And of course, some enzymes, eg trypsin, do not discriminate between R and 
K (at P1). Others however _DO_ discriminate.  I don't at what level you've 
looked at the crystal structure - but discrimination can depend on subtle 
features of the geometry of the enzyme subsite and the position of the D/E 
within in it.

  In other words, if you're looking at the structure and saying, "well there's 
the subsite, and there's a negative charge at the bottom," then that's not close
to being enough information to form an opinion as to whether K or R or either
derivatives should be recognised.

  Hope this helps a bit...

  Regards,

-- Simon
_____________________________________________________________________________
|
|  ,_ o     Simon M. Brocklehurst,
| /  //\,   Oxford Centre for Molecular Sciences, Department of Biochemistry,
|   \>> |   University of Oxford, Oxford, UK.
|    \\,    E-mail: smb at bioch.ox.ac.uk | WWW: http://www.ocms.ox.ac.uk/~smb/ 
|____________________________________________________________________________