The contribution of N-terminal region residues of cystatin A (stefin A) to the affinity and kinetics of inhibition of papain, cathepsin B, and cathepsin L.
The affinity and kinetics of binding of three N-terminally truncated variants of the cysteine proteinase inhibitor cystatin A to cysteine proteinases were characterized. Deletion of Met-1 only minimally altered the inhibitory properties of the protein. However, deletion also of Ile-2 resulted in reduced affinities of 900-, >/=3-, and 200-fold for papain and cathepsins L and B, respectively. Further truncation of Pro-3 substantially increased the inhibition constants to approximately 0.5 microM for papain and cathepsin L and to 60 microM for cathepsin B, reflecting additionally 2 x 10(3)-, 2 x 10(4)-, and 400-fold decreased affinities, respectively. The reductions in affinity shown by the latter mutant indicate that the N-terminal region contributes about 40% of the total free energy of binding of cystatin A to cysteine proteinases. Moreover, Pro-3 and to a lesser extent Ile-2 are the residues responsible for this binding energy. The reduced affinities for papain and cathepsin L were due only to higher dissociation rate constants, whereas both lower association and higher dissociation rate constants contributed to the decreased affinity for cathepsin B. These differential effects indicate that the N-terminal portion of cystatin A primarily functions by stabilizing the complexes with enzymes having easily accessible active-site clefts, e.g., papain and cathepsin L. In contrast, the N-terminal region is required also for an initial binding of cystatin A to cathepsin B, presumably by promoting the displacement of the occluding loop and allowing facile interaction of the rest of the inhibiting wedge with the active-site cleft of the enzyme.
Estrada
S
S
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Center.
Pavlova
A
A
Björk
I
I
D000818:Animals; D002401:Cathepsin B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002942:Circular Dichroism; D015891:Cystatins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D003546:Cysteine Endopeptidases; D010450:Endopeptidases; D005822:Genetic Vectors / Q000138:chemical synthesis; D006801:Humans; D007700:Kinetics; D010206:Papain / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D011485:Protein Binding / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017384:Sequence Deletion; D012756:Sheep
D015891:Cystatins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L; D010206:Papain