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Intrinsic Evolutionary Constraints on Protease Structure, Enzyme

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Intrinsic evolutionary constraints on protease PNAS PLUS structure, enzyme acylation, and the identity of the catalytic triad Andrew R. Buller and Craig A. Townsend1 Departments of Biophysics and Chemistry, The Johns Hopkins University, Baltimore MD 21218 Edited by David Baker, University of Washington, Seattle, WA, and approved January 11, 2013 (received for review December 6, 2012) The study of proteolysis lies at the heart of our understanding of enzyme evolution remain unanswered. Because evolution oper- biocatalysis, enzyme evolution, and drug development. To un- ates through random forces, rationalizing why a particular out- derstand the degree of natural variation in protease active sites, come occurs is a difficult challenge. For example, the hydroxyl we systematically evaluated simple active site features from all nucleophile of a Ser protease was swapped for the thiol of Cys at serine, cysteine and threonine proteases of independent lineage. least twice in evolutionary history (9). However, there is not This convergent evolutionary analysis revealed several interre- a single example of Thr naturally substituting for Ser in the lated and previously unrecognized relationships. The reactive protease catalytic triad, despite its greater chemical similarity rotamer of the nucleophile determines which neighboring amide (9). Instead, the Thr proteases generate their N-terminal nu- can be used in the local oxyanion hole. Each rotamer–oxyanion cleophile through a posttranslational modification: cis-autopro- hole combination limits the location of the moiety facilitating pro- teolysis (10, 11). These facts constitute clear evidence that there ton transfer and, combined together, fixes the stereochemistry of is a strong selective pressure against Thr in the catalytic triad that catalysis. All proteases that use an acyl-enzyme mechanism natu- is somehow relieved by cis-autoproteolysis. Because the catalytic rally divide into two classes according to which face of the peptide triad is the principal system through which enzymatic rate ac- substrate is attacked during catalysis. We show that each class is celeration has been studied, the inability to rationalize the evo- subject to unique structural constraints that have governed the lutionary selection of the catalytic nucleophile is a fundamental BIOCHEMISTRY convergent evolution of enzyme structure. Using this framework, weakness in our understanding of biocatalysis. Motivated by this we show that the γ-methyl of Thr causes an intrinsic steric clash simple, yet unexplained structure–function relationship, we sought that precludes its use as the nucleophile in the traditional catalytic to answer the question: How do the chemical and structural con- triad. This constraint is released upon autoproteolysis and we pro- straints of proteolysis combine to shape the evolution of a protease pose a molecular basis for the increased enzymatic efficiency in- active site? troduced by the γ-methyl of Thr. Finally, we identify several Two strategies can be undertaken to probe the properties of classes of natural products whose mode of action is sensitive to diverse catalytic architectures. The first is the contemporary ex- the division according to the face of attack identified here. This perimental approach whereby all parameters of interest in an analysis of protease structure and function unifies 50 y of bioca- active site are systematically varied and analyzed in silico for talysis research, providing a framework for the continued study of catalytic viability. This technique was used by Smith et al. to show enzyme evolution and the development of inhibitors with in- that the catalytic triad of esterases adopts a consensus geometry creased selectivity. that minimizes reorganization during the multistep mechanism (12). Computational explorations are unsurpassed in their detail peptidase | antibiotic stereochemistry | N-terminal nucleophile and broad applicability, but hindered by their need to explicitly anticipate the structural and mechanistic diversity generated he acceleration of chemical reactions is essential to all bio- through natural selection. We elected to pursue an alternative Tchemistry. The generation of reactive species by enzymes is strategy to elucidate the constraints that guide active site evo- tightly controlled and limited by the chemical makeup of the 20 proteinogenic amino acids. In isolation, no protein residue har- Significance bors a strong nucleophile at physiological pH. Rather, enzymatic activity arises after protein folding introduces cooperative in- The structure–function relationship of proteases is central to teractions that selectively amplify the reactivity of their otherwise our understanding of biochemistry. Nature has evolved at least weakly active functional groups. The preeminent system through 23 independent solutions to this problem, using an acylation which this phenomenon has been studied is the Ser-His-Asp mechanism. We examined the structures of these proteases, catalytic triad of the Ser proteases (1). Several distinct molecular using a new framework to characterize the geometric rela- strategies have been identified that contribute to rate enhance- tionships within each active site. This analysis revealed the ment. Substrate binding increases the local concentration of orientation of the base determines the stereochemistry of ca- interacting species, general acids and bases facilitate proton talysis and elucidated why threonine does not substitute for transfer, and a high-energy step can be split into multiple lower- serine in the catalytic triad. These observations explain how energy steps (2). These distinct molecular strategies may be the absolute stereostructures of natural protease inhibitors unified through the formalism of transition-state theory (3) and prevent off-target inhibition and serve as boundary conditions the idea that the active site is electrostatically preorganized for to enzyme design. transition-state stabilization (4, 5). Although the biophysics of rate acceleration are intricate and sensitive to even minor struc- Author contributions: A.R.B. and C.A.T. designed research; A.R.B. performed research; tural perturbations, evolution has converged on a catalytic triad A.R.B. and C.A.T. analyzed data; and A.R.B. and C.A.T. wrote the paper. (or diad) with a reactive Ser, Cys, or Thr nucleophile more than The authors declare no conflict of interest. 25 separate times to facilitate central biochemical reactions such This article is a PNAS Direct Submission. as hydrolysis (6), transacylation (7), and phosphorylation (8). 1To whom correspondence should be addressed. E-mail: [email protected]. Our understanding of biochemistry has progressed into an This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. increasingly complex picture but many basic questions about 1073/pnas.1221050110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1221050110 PNAS | Published online February 4, 2013 | E653–E661 Downloaded by guest on September 27, 2021 lution that is not computational, but observational. Studying the the energetics of hydrolysis are different from enzyme acylation, large set of naturally occurring structures limits analyses to coarser they share many structural constraints. As is shown in this in- features, but greatly increases the universality of the results. This vestigation, it is sufficient to consider only the acylation step of the technique requires only a diverse set of enzyme structures and reaction, as displayed in Fig. 1, to rationalize much of the ob- a rudimentary understanding of organic and biochemistry. By servable convergent evolution of proteases. recognizing the constraints imposed by bond geometries and Evolution has selected for many different residues to activate steric clashes, it is easy to compare the potential catalytic archi- the nucleophile, but the cooperative interactions that engender tectures for a given reaction. reactivity are effectively “tuned” differently for each individual Results from the veritable explosion of gene sequencing and enzyme. This principle was first tested by the chemical swapping protein structure determination have identified at least 23 folds of the hydroxyl nucleophile of subtilisin for a thiol and observing related through convergent evolution that support proteolysis loss of proteolytic activity (16, 17). More exhaustive studies using through an acyl-enzyme intermediate (9). We cross-compared site-directed mutagenesis of both subtilisin and trypsin showed the active site architecture among these “analogous” enzymes that alteration of any component of the catalytic triad results in (13), each of which has been optimized for catalysis through a large decrease in enzyme efficiency (18, 19). Interestingly, the → natural selection, to interrogate the entire known structural di- Ser Thr variant of trypsin created a worse enzyme than simul- versity that supports efficient proteolysis. We first identified key taneously mutating all three residues of the catalytic triad. parameters that define active site architecture, such as the ori- Conversely, when the N-terminal Thr nucleophile of the pro- β entation of substrate binding, the φ, ψ angles of the nucleophile, teasome -subunit was mutated to Ser, there was a reduction in and the stereochemistry of catalysis. Analysis of all Ser, Cys, and its catalytic power (20). The juxtaposition of these results and the γ Thr proteases of independent lineage showed they were all inability to rationalize the opposite effects
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