Björk, Ingemar
- Institutionen för molekylär biovetenskap, Sveriges lantbruksuniversitet
Sammanfattning
Replacement of the three N-terminal residues preceding the conserved Gly of cystatin A by the corresponding 10-residue long segment of cystatin C increased the affinity of the inhibitor for the major lysosomal cysteine proteinase, cathepsin B, by similar to15-fold. This tighter binding was predominantly due to a higher overall association rate constant. Characterization of the interaction with an inactive Cys29 to Ala variant of cathepsin B indicated that the higher rate constant was a result of an increased ability of the N-terminal region of the chimeric inhibitor to promote displacement of the cathepsin B occluding loop in the second binding step. The low dissociation rate constant for the binding of cystatin A to cathepsin B was retained by the chimeric inhibitor, which therefore had a higher affinity for this enzyme than any natural cystatin identified so far. In contrast, the N-terminal substitution negligibly affected the ability of cystatin A to inhibit papain. However, substitutions of Gly75 in the second binding loop of cystatin A by Trp or His, making the loop similar to those of cystatins C or B, respectively, increased the affinity for papain by similar to10-fold. This enhanced affinity was due to both a higher association rate constant and a lower dissociation rate constant. Modeling of complexes between the two variants and papain indicated the possibility of favorable interactions being established between the substituting residues and the enzyme. The second-loop substitutions negligibly affected or moderately reduced the affinity for cathepsin B. Together, these results show that the inhibitory ability of cystatins can be substantially improved by protein engineering.
Publicerad i
Biochemistry
2003, volym: 42, nummer: 38, sidor: 11326-11333
Utgivare: AMER CHEMICAL SOC
SLU författare
UKÄ forskningsämne
Läkemedelskemi
Publikationens identifierare
- DOI: https://doi.org/10.1021/bi030119v
Permanent länk till denna sida (URI)
https://res.slu.se/id/publ/1086