Autocatalytic processing of recombinant human procathepsin B is a bimolecular process

Abstract

Cathepsin B and other lysosomal cysteine proteinases are synthesized as inactive zymogens, which are converted to their mature forms by other proteases or by autocatalytic processing. Procathepsin B autoactivation was shown in vitro at pH 4.5 to be a bimolecular process with K s and k cat values of 2.1 þ 0.9 W WM and 0.12 þ 0.02 s 31 , respectively. Autoactivation is substantially accelerated in the presence of active cathepsin B molecules, indicating that mature cathepsin B is the catalytic species in the process. Proenzyme is cleaved without significant conformational changes as judged by circular dichroism, suggesting that propeptide unfolding occurs only after the cleavage. Procathepsin B autoactivation is pH-dependent with a pH optimum at 4.5 and with no processing observed at pH s 6.0. However, in the presence of 0.5 W Wg/ml of dextran sulfate, relatively rapid processing is observed even at pH 6.5 (t 1a2 V V90 min), suggesting that glycosaminoglycans are involved in in vivo processing of lysosomal cysteine proteases.z 1999 Federation of European Biochemical Societies.

Glycosaminoglycans Modulate Activation, Activity, and Stability of Tripeptidyl-peptidase I in Vitro and in Vivo

“…As we have evidenced, polyanionic compounds not only are able to markedly enhance the yields and rates of pro-TPP activation, but what may be especially important, they also are extending pro-TPP I activation toward less acidic pH, bringing the feasibility of pro-TPP I autoactivation within the physiological pH range of lysosomes. Similar stimulatory effect of GAGs also was reported for two lysosomal cysteine proteases, pro-cathepsins B and L (34,35,49). Interestingly, congopain, the major cysteine protease from Trypanosoma congolense, was activated in the presence of DS, even at a basic pH as high as 8.0 (33).…”

“…However, no conformation changes of pro-cathepsin L during autoprocessing could be found by further studies (66), and similarly, we could not detect any major conformation changes during pro-TPP I autoactivation (25). Therefore, the mechanism proposed later on, implying that DS and related sulfated GAGs can substantially accelerate in vitro autoactivation of lysosomal proteases by weakening the interaction between the propeptide and the catalytic part (35) appears to be also relevant to the interactions of GAGs with TPP I. During the activation, the binding of polyanions to the mature portion of the pro-TPP I might interrupt the interaction of the prosegment with its cognate prote- FIG.…”

“…The Effect of GAGs on Pro-TPP I Maturation in Vitro-It was demonstrated that negatively charged compounds, such as dextran sulfate (DS), are able to accelerate the processing of several lysosomal enzymes including cathepsins B and L (34,35).…”

Cathepsin L1, the Major Protease Involved in Liver Fluke (Fasciola hepatica) Virulence

“…As the stability of the propeptide-protease complex is dependent on electrostatic interactions, reduction of the environmental pH, weakens the bond between the propeptide and the catalytic site. As a consequence, the proenzyme possibly adopts a looser conformation, in which the propeptide is bound less tightly into the active site making it more susceptible to proteolysis (16,27). The precise mechanism of proteolytic conversion from proenzyme to mature enzyme is still actively debated.…”

“…The threedimensional structure of procathepsin L (14) and procathepsin B (28,29,30) show that the N terminus of the mature enzyme is quite removed from the active site thus making it difficult to visualize an autocatalytic cleavage event. Moreover, circular dichroism studies reveal that activation does not involve significant conformational changes in the structure of procathepsin L (15) or procathepsin B (16). Therefore, the initial event in autocatalysis may involve an active proenzyme, possibly created by the reduced pH, that cleaves another proenzyme in the vicinity of the N terminus and sets off a chain reaction (15,16).…”