Open access publications in the SLU publication database

Abstract

Cystatin A, a mammalian cysteine proteinase inhibitor, was expressed in a bacterial system. The purified protein was fully active. It inhibited the proteinases,papain and cathepsin L, strongly, with K[ values of 0.2-20 pM, whereas the affinities for actinidin and cathepsins B, C and H were in the range of 1-40 nM. The binding to papain and cathepsin L was rapid, with &ass -3-5 x 10^ M '1 s '1, whereas the inhibition of cathepsin B was -100-fold slower. The binding to papain and consistent with a one-step binding mechanism.Gly-4 mutants of cystatin A had lower affinities for papain and cathepsins B and L than the wild-type inhibitor. In general, the K\ values increased with the size of the side chain of the mutant. Even the smallest mutation, Gly-4 to Ala, had substantial effects, and the charged Gly-4 to Glu or Arg variants had affinities for the enzymes that were more than five orders of magnitude lower than those of the wild-type. The rate of binding of the mutants to papain and cathepsin L was unaffected, but was lower than that of the wildtype for cathepsin B, presumably reflecting structural differences in the enzymes.Sequential truncations of the N-terminal region of cystatin A showed that Ile-2 and, to an even greater extent, Pro-3 were the residues of the this region that contributed to the binding of the inhibitor to papain and cathepsins B and L. In contrast with the primarily anchoring role of the N-terminal region of cystatin A to papain and cathepsin L, this region was needed also for mantaining the rate of association of the inhibitor with cathepsin B.A fluorescent probe was linked to a recombinant cystatin A variant via an extra Nterminal cysteine. The labelled inhibitor had eight-fold higher affinity for papain than the wild-type cystatin, due to an increased association rate constant. The binding of the Nterminal label followed saturation kinetics, indicating that the N-terminal region of the labelled cystatin binds to the enzyme in the second step of a two-step reaction mechanism.