20 March, 2002
ABSTRACT: An Escherichia coli overexpression system for the 20S proteasome from the archaebacteria Thermoplasma acidophilum was obtained and purified recombinant enzyme was used to understand the mechanism of activation of the enzyme. Known activators include magnesium and other salts, and SDS and similar detergents. Two previously unknown activators, buffer ions and some proteins, have also been identified and their points of activation in the mechanism isolated.
Using the kinetics of a slow binding inhibitor, Z-LLLal, and measuring the remaining activity with the substrate Suc-LLVY-AMC, the steps affected by these activators have been identified. SDS, previously thought to enhance the rate of association between the enzyme and substrate, has been found to enhance the rate constant for release of product. Magnesium effects have been found to be largely due to ionic strength, and are due mainly to the stabilization of the tetrahedral intermediate. The novel activator buffer ions have been found to promote protonation of the first leaving group of the newly formed product, and are pH and p$K_a$ sensitive.
Secondly, evidence has been found that the 20S proteasome core particle can digest destabilized proteins at a biologically appreciable rate, similar to that of the proteases trypsin and chymotrypsin. This rate is significantly faster than the rate of digestion of the peptide Suc-LLVY-AMC and is due to the association of 14 active sites in close proximity. Only destabilized proteins can be digested with any significant rate, as they must be able to enter the proteasome active site chamber. It is this step, substrate entry into the active site chamber, which has become the rate limiting step.
Lastly, cooperativity effects have been observed in the proteasome. Homotropic cooperativity has been observed in the presence of subsaturating amounts of the inhibitor peptide. Negative cooperativity has been observed with high substrate concentrations, leading to a depression of proteasome activity by approximately 30\%.
These findings illustrate several key facets of the proteasome mechanism, and suggest an interlinked energy landscape for the hydrolysis reaction. Several suggestions for future experiments are also provided.
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