In MEROPS, an online database that provides an insight into peptidases, there are 16 clans of cysteine peptidases and some that are unclassified, of which four among them include proteases with mixed catalytic types. Ĭysteine proteases use the reactive site cysteine as the catalytic nucleophile and the histidine to perform peptide bond hydrolysis. Because no review, including ours, completely covers a broad topic such as protein inhibitors of cysteine proteases, we want to remind readers that other related reviews have been undertaken that describe various aspects of cysteine protease inhibition. This link between biochemical principles of inhibition and physiology does not require an update, however, the determination of a number of new structures of cysteine protease inhibitors indicates that canonical mechanisms of inhibition should be updated. To gain insight into the relative differences between various protease inhibitor interactions, we rely predominantly on their ratio, Ki. Detailed kinetic studies in which K ass and K diss are measured are seldomly performed. The delay-type inhibitors irreversibly (or pseudo-irreversibly) and slowly bind their target, thereby enabling proteolysis for a limited amount of time, whereas pro-inhibitors require initial processing by a protease to become inhibitory. The buffer-type inhibitors reversibly and rapidly bind proteases, and when their physiological substrate appears, they also rapidly release them and thereby prevent undesired and potentially harmful proteolysis in the absence of their substrate. The threshold-type inhibitors prevent undesired protease activation. They can be further divided into threshold, buffer, delay, and pro-inhibitor sub-types. Regulatory inhibitors, by comparison, modulate the protease activity under physiological conditions. Emergency inhibitors rapidly trap a protease and maintain it in a stable complex preventing any undesired activity. Two types of inhibitors were introduced, emergency and regulatory. This suggest that inhibitors were of physiological relevance when the delay time was below 1 s and, for reversible inhibitors, when the stability time was above 10 min. The stability time of reversible inhibitors is defined as the minimal time in which the EI (E, enzyme I, inhibitor) complex remains undissociated (t(s) = ln 2/k diss), where k diss represents the dissociation rate constant. In this equation, I o represents the physiological concentration of the inhibitor and k ass is an approximation of the association rate constant. Delay time, d(t), is the time needed to achieve ≈99% of inhibition (d(t) = ln 2/I o × k ass), and roughly equals seven half-lives of the reaction. In the review, we classified the inhibitors according to their physiological roles using quantitative criteria of enzyme kinetics, the delay time of inhibition, and the stability time of inhibition, established by Joseph Bieth in the 1980s.
Previously, we reviewed cysteine protease protein inhibitors and their role in regulation of proteolysis.
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Finally, we show a number of examples and provide hints on how to engineer protein inhibitors. It appears that all proteases, with the exception of papain-like proteases, belong to the first group of proteases. In contrast, when the S1 binding site is in part exposed to solvent, the substrate-like inhibition cannot be employed.
When the S1 binding site is shaped as a pocket buried in the structure of protease, inhibitors can apply substrate-like binding mechanisms. However, the analysis suggests that the shape of the active site cleft of proteases imposes some restraints. The structural data make it clear that the “lock and key” mechanism is a historical concept with limited validity.
In addition, there appears to be no general rule governing the inhibitory mechanism. It appears that nature can convert almost any starting fold into an inhibitor of a protease. The gathered structural data indicate that the protein fold is not a major obstacle for the evolution of a protease inhibitor. In this review, we analyzed the mechanisms of inhibition of cysteine proteases on the basis of structural information and compiled kinetic data.
Protein inhibitors of proteases are an important tool of nature to regulate and control proteolysis in living organisms under physiological and pathological conditions.
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