Intrinsic evolutionary constraints on protease structure, enzyme acylation, and the identity of the catalytic triad

Abstract

The study of proteolysis lies at the heart of our understanding of biocatalysis, enzyme evolution, and drug development. To understand the degree of natural variation in protease active sites, we systematically evaluated simple active site features from all serine, cysteine and threonine proteases of independent lineage. This convergent evolutionary analysis revealed several interrelated and previously unrecognized relationships. The reactive rotamer of the nucleophile determines which neighboring amide can be used in the local oxyanion hole. Each rotamer-oxyanion hole combination limits the location of the moiety facilitating proton transfer and, combined together, fixes the stereochemistry of catalysis. All proteases that use an acyl-enzyme mechanism naturally divide into two classes according to which face of the peptide substrate is attacked during catalysis. We show that each class is subject to unique structural constraints that have governed the convergent evolution of enzyme structure. Using this framework, we show that the γ-methyl of Thr causes an intrinsic steric clash that precludes its use as the nucleophile in the traditional catalytic triad. This constraint is released upon autoproteolysis and we propose a molecular basis for the increased enzymatic efficiency introduced by the γ-methyl of Thr. Finally, we identify several classes of natural products whose mode of action is sensitive to the division according to the face of attack identified here. This analysis of protease structure and function unifies 50 y of biocatalysis research, providing a framework for the continued study of enzyme evolution and the development of inhibitors with increased selectivity.peptidase | antibiotic stereochemistry | N-terminal nucleophile T he acceleration of chemical reactions is essential to all biochemistry. The generation of reactive species by enzymes is tightly controlled and limited by the chemical makeup of the 20 proteinogenic amino acids. In isolation, no protein residue harbors a strong nucleophile at physiological pH. Rather, enzymatic activity arises after protein folding introduces cooperative interactions that selectively amplify the reactivity of their otherwise weakly active functional groups. The preeminent system through which this phenomenon has been studied is the Ser-His-Asp catalytic triad of the Ser proteases (1). Several distinct molecular strategies have been identified that contribute to rate enhancement. Substrate binding increases the local concentration of interacting species, general acids and bases facilitate proton transfer, and a high-energy step can be split into multiple lowerenergy steps (2). These distinct molecular strategies may be unified through the formalism of transition-state theory (3) and the idea that the active site is electrostatically preorganized for transition-state stabilization (4, 5). Although the biophysics of rate acceleration are intricate and sensitive to even minor structural perturbations, evolution has converged on a catalytic triad (or diad) with a rea...

Structure of the Catalytic Domain of the Class I Polyhydroxybutyrate Synthase from Cupriavidus necator

“…However, mutation of Asp 480 to Asn in CnPhaC was shown to result in a 50-fold decrease in the rate of enzyme acylation, suggesting that Asp 480 could play a role in nucleophilic activation, although mutation of the equivalent residue in the class III synthase from A. vinosum had no effect on the acylation rate (8,9). We note that it is also possible that the resting state of the enzyme is zwitterionic, containing a preformed cysteine thiolate and histidine imidazolium pair, as has been demonstrated in cysteine proteases (40,41). In either case, the histidine base is (re)generated through donation of the imidazolium proton to the CoASH leaving group.…”

Elucidation of the Specific Function of the Conserved Threonine Triad Responsible for Human l-Asparaginase Autocleavage and Substrate Hydrolysis

“…A recent analysis of serine and cysteine proteases clarifies that these catalytic residues cannot be replaced by threonine due to the steric constraints imposed by the threonine γ-methyl group [24]. However, these constraints are eliminated in Ntn enzymes, explaining the compatibility of threonine (though Ntn enzymes also use serine and cysteine).…”

“…In the case of the proteasome β-subunit, the first member of “Thr proteases”, replacing the threonine by a serine reduces the reaction rate [25]. This was explained by the γ-methyl group of the threonine favoring the reactive threonine rotamer [24]. Likewise, for the Flavobacterium glycosylasparaginase, an enzyme very similar to hASNase3, mutation of the catalytic threonine to either cysteine or serine resulted in reduced but measurable enzymatic activity [26].…”

Structural Insights into an Oxalate-producing Serine Hydrolase with an Unusual Oxyanion Hole and Additional Lyase Activity

“…From a chemical structure perspective, C2-C3 bond cleavage accounts for oxalate production, and C4-S bond cleavage is responsible for the release of acetoacetate and CoA. It is highly evident that within the C-domain crevice, the catalytic triad is responsible for C2-C3 bond cleavage in a manner identical to other serine hydrolases that cleave C-C bonds between trigonal and tetrahedral carbon atoms (18,23). Specifically, given that C2 in the C6-CoA adduct is the only sp 2 carbon, Ser-935, upon activation by His-1069, performs nucleophilic attack on the C2 carbonyl carbon, forming a tetrahedral reaction intermediate (Fig.…”