The interaction of human recombinant full-length cathepsin S propeptide (amino acids 16-114) with mature cysteine proteinases was studied with respect to selectivity and pH dependence. The inhibitory capacity was tested towards mature human recombinant cathepsin S, purified cathepsin L from rat and Paramecium tetraurelia, rat cathepsin B, human cathepsin H, and papain. The propeptide of cathepsin S strongly inhibited cathepsin S (Ki = 0.27 nM) and the two cathepsin L species (Ki = 0.36 nM) at neutral pH. Papain, and to a minor extent cathepsin H, hydrolyzed the propeptide of cathepsin S, leading to competition with the hydrolysis of the fluorogenic substrates in the respective assays. Cathepsin B activity was nearly unaffected up to micromolar propeptide concentrations in the assay. The inhibition of cathepsin-L-like peptidases was diminished with decreasing pH, probably due to dramatic changes in the conformation of the propeptide. This assumption was supported by far-ultraviolet CD spectroscopy and by the finding of rapid hydrolysis of the cathepsin S propeptide by cathepsin L at pH values less than 5.5.

Cancer cell energy metabolism is characterized by a high glycolytic rate, which is maintained under aerobic conditions. In Ehrlich ascites tumour cells, the concentration of fructose 2,6-bisphosphate (Fru-2,6-P2), the powerful activator of 6-phosphofructo-1-kinase, is tenfold increased. The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), synthesizing and degrading Fru-2,6-P2, was characterized. The molecular mass is 120 kDa. The dependence of PFK-2 activity on the substrate concentrations is hyperbolic (Km for Fru-6-P = 0.09 mM; Km for ATP = 0.7 mM), while the dependence of the FBPase-2 activity on the concentrations of Fru-2,6-P2 is sigmoidal (K0.5 for Fru-2,6-P2 = 4 microM). The PFK-2/FBPase-2 activity ratio is 1. PFK-2 activity is inhibited by citrate (I0.5 = 0.17 mM) and phosphoenolpyruvate (I0.5 = 0.08 mM) but only weakly by glycerol 3-phosphate (I0.5 = 1.57 mM). In contrast to the liver enzyme, the activity of tumour PFK-2/FBPase-2 is not influenced by the action of cAMP-dependent protein kinase. The kinetic properties as well as ion-exchange chromatography pattern differ from their normal counterparts in liver and muscle. The properties are likely to contribute to the maintenance of the high glycolytic rate in these tumour cells.

Cathepsin L-like cysteine proteinases contain an evolutionarily highly conserved a-helical motif in the proregion. This is called the ER(F/W)N(I/V)N motif according to the conserved amino acids along one side of the helix. We studied the function of this motif using site-directed mutagenesis experiments of human procathepsin S. We replaced each of these amino acids with alanine and constructed deletion mutants lacking parts of the helix. All mutants were expressed in HEK 293 cells, but only one, W52A, was not processed to mature cathepsin S, nor was it phosphorylated or secreted into the culture medium. W52 is part of the hydrophobic core in the propeptide region of cathepsin S comprising two additional tryptophan residues, W28 and W31, also conserved among cathepsin L-like cysteine peptidases. Replacement of the latter with alanine led to consequences similar to those with the W52A mutation. Recombinant propeptides containing mutations of one of the three tryptophan residues were three orders of magnitude less effective as inhibitors of mature cathepsin S than the wild-type propeptide. The results point to a dominant role of the respective hydrophobic stack in the proper folding, transport and maturation of procathepsin S and related cathepsin L-like cysteine proteinases.

Cathepsin L-like cysteine proteinases contain an evolutionarily highly conserved a-helical motif in the proregion. This is called the ER(F/W)N(I/V)N motif according to the conserved amino acids along one side of the helix. We studied the function of this motif using site-directed mutagenesis experiments of human procathepsin S. We replaced each of these amino acids with alanine and constructed deletion mutants lacking parts of the helix. All mutants were expressed in HEK 293 cells, but only one, W52A, was not processed to mature cathepsin S, nor was it phosphorylated or secreted into the culture medium. W52 is part of the hydrophobic core in the propeptide region of cathepsin S comprising two additional tryptophan residues, W28 and W31, also conserved among cathepsin L-like cysteine peptidases. Replacement of the latter with alanine led to consequences similar to those with the W52A mutation. Recombinant propeptides containing mutations of one of the three tryptophan residues were three orders of magnitude less effective as inhibitors of mature cathepsin S than the wild-type propeptide. The results point to a dominant role of the respective hydrophobic stack in the proper folding, transport and maturation of procathepsin S and related cathepsin L-like cysteine proteinases.

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