Papain-like cysteine proteinases. Regulation by proteinase inhibitors and pH

A low-molecular-weight protein inhibitor of cysteine proteinases from bovine skin was sequenced and identified as bovine stefin A. Kinetic analyses showed that it bound tightly and rapidly to the lysosomal cystene proteinases, cathepsins H and L, but more slowly, although still tightly, to cathepsin B. Stefin A in skin is presumably involved in the control of endogenous cysteine proteinases and in protection against invasive organisms that use cysteine proteinases to penetrate the skin. Intact L- and H-kininogens bound simultaneously two molecules of cysteine proteinases, showing that both inhibitory kininogen domains are active also in the intact proteins. The two sites bound proteinases with different rates and affinities and did not interfere with each other's binding ability. The kininogen light chains, as well as binding of H-kininogen to surfaces, did not affect proteinase binding. Under most conditions, proteinases presumably bind only to the faster and tighter binding site of the kininogens, whereas the other site remains largely unoccupied. The functional role of the tandem arrangement of proteinase binding sites is thus unclear. Exposure of cathepsin L to pH below apprx 4 resulted in an irreversible loss of enzyme activity. This loss was accompanied by a major conformational change of the protein, as evident by marked circular dichroism changes and a high activation energy. The process was dependent on substrate concentration in a way suggesting that inactivation starts at the active site. Cathepsin L, and presumably also other lysosomal cysteine proteinases, thus can be spontaneously inactivated at the low pH in lysosomes, probably resulting in short lifetimes. Papain, a model for lysosomal cysteine proteinases, was irreversibly inactivated at both alkaline and acid pH. The process was accompanied by a loss of structure, initiated in the active-site region. However, the rate of the inactivation was probably regulated by a cluster of charged residues remote from the active site. This inactivation mecahnism may be common for all papain-like cysteine proteinases, although the distribution and number of charged residues in individual enzymes result in different stabilities.