The proteolytic enzyme stromelysin-1 is a member of the family of matrix metalloproteinases and is believed to play a role in pathological conditions such as arthritis and tumor invasion. Stromelysin-1 is synthesized as a proenzyme that is activated by removal of an N-terminal prodomain. The active enzyme contains a catalytic domain and a C-terminal hemopexin domain believed to participate in macromolecular substrate recognition. We have determined the three-dimensional structures of both a C-truncated form of the proenzyme and an inhibited complex of the catalytic domain by X-ray diffraction analysis. The catalytic core is very similar in the two forms and is similar to the homologous domain in fibroblast and neutrophil collagenases, as well as to the stromelysin structure determined by NMR. The prodomain is a separate folding unit containing three a-helices and an extended peptide that lies in the active site of the enzyme. Surprisingly, the amino-to-carboxyl direction of this peptide chain is opposite to that adopted by the inhibitor and by previously reported inhibitors of collagenase. Comparison of the active site of stromelysin with that of thermolysin reveals that most of the residues proposed to play significant roles in the enzymatic mechanism of thermolysin have equivalents in stromelysin, but that three residues implicated in the catalytic mechanism of thermolysin are not represented in stromelysin.
The field of biochemistry is currently faced with the enormous challenge of assigning functional significance to more than thirty thousand predicted protein products encoded by the human genome. In order to accomplish this daunting task, methods will be required that facilitate the global analysis of proteins in complex biological systems. Recently, methods have been described for simultaneously monitoring the activity of multiple enzymes in crude proteomes based on their reactivity with tagged chemical probes. These activity based probes (ABPs) have used either radiochemical or biotin/avidin-based detection methods to allow consolidated visualization of numerous enzyme activities. Here we report the synthesis and evaluation of fluorescent activity based probes for the serine hydrolase super-family of enzymes. The fluorescent methods detailed herein provide superior throughput, sensitivity, and quantitative accuracy when compared to previously described ABPs, and provide a straight-forward platform for high-throughput proteome analysis.
The kinetic and thermodynamic features of reactions catalyzed by present-day enzymes appear to be the consequence of the evolution of these proteins toward maximal catalytic effectiveness. These features are identified and analyzed (in detail for one substrate-one product enzymes) by using ideas that link the energetics of the reaction catalyzed by an enzyme to the maximization of its catalytic efficiency. A catalytically optimized enzyme will have a value for the "internal" equilibrium constant (Kint, the equilibrium constant between the substrates and the products of the enzyme when all are bound productively) that depends on how close to equilibrium the enzyme maintains its reaction in vivo. Two classes are apparent. For an enzyme that operates near equilibrium, the catalytic efficiency is sensitive to the value of Kint, and the optimum value of Kint is near unity. For an enzyme that operates far from equilibrium, the catalytic efficiency is less sensitive to the value of Kint, and Kint assumes a value that ensures that the rate of the chemical transformation is equal to the rate of product release. In each of these cases, the internal thermodynamics is "dynamically matched", where the concentrations of substrate- and product-containing complexes are equal at the steady state in vivo.
Genome sequencing projects have uncovered many novel enzymes and enzyme classes for which knowledge of active site structure and mechanism is limited. To facilitate mechanistic investigations of the numerous enzymes encoded by prokaryotic and eukaryotic genomes, new methods are needed to analyze enzyme function in samples of high biocomplexity. Here, we describe a general strategy for profiling enzyme active sites in whole proteomes that utilizes activity-based chemical probes coupled with a gel-free analysis platform. We apply this gel-free strategy to identify the sites of labeling on enzymes targeted by sulfonate ester probes. For each enzyme examined, probe labeling was found to occur on a conserved active site residue, including catalytic nucleophiles (e.g., C32 in glutathione S-transferase omega) and bases/acids (e.g., E269 in aldehyde dehydrogenase-1; D204 in enoyl CoA hydratase-1), as well as residues of unknown function (e.g., D127 in 3 beta-hydroxysteroid dehydrogenase/isomerase-1). These results reveal that sulfonate ester probes are remarkably versatile activity-based profiling reagents capable of labeling a diversity of catalytic residues in a range of mechanistically distinct enzymes. More generally, the gel-free strategy described herein, by consolidating into a single step the identification of both protein targets of activity-based probes and the specific residues labeled by these reagents, provides a novel platform in which the proteomic comparison of enzymes can be accomplished in unison with a mechanistic analysis of their active sites.
Characterization and functional annotation of the large number of proteins predicted from genome sequencing projects poses a major scientific challenge. Whereas several proteomics techniques have been developed to quantify the abundance of proteins, these methods provide little information regarding protein function. Here, we present a gel-free platform that permits ultrasensitive, quantitative, and high-resolution analyses of protein activities in proteomes, including highly problematic samples such as undiluted plasma. We demonstrate the value of this platform for the discovery of both disease-related enzyme activities and specific inhibitors that target these proteins.capillary electrophoresis ͉ fluorescence ͉ MS ͉ protease T he completion of several major genome sequencing projects has both accelerated the pace and broadened the scale of modern biology. For example, it is estimated that a complete molecular understanding of the human organism will require the characterization of at least 100,000 proteins (1). Several innovations in the field of proteomics have advanced to meet the demands of postgenome biology. Particularly, improvements in the resolving capacity and reproducibility of 2D gel electrophoresis (2) and the increased sensitivity and accuracy of modern MS methods (3) have enabled the global characterization of protein expression levels in complex biological systems. Nonetheless, the large dynamic range of protein expression in biological tissues and fluids renders these methods ineffective at detecting and͞or quantitating low-abundance proteins in the absence of labor-intensive prefractionation or enrichment procedures (2, 4). Moreover, abundance-based proteomic strategies fail to provide direct information regarding protein function or activity.To address these limitations, a chemical proteomic technology called activity-based protein profiling (ABPP) (5) has emerged that utilizes active site-directed probes to enable the parallel measurement of the activity of many enzymes in complex proteomic mixtures (5-8). Activity-based probes (ABPs) enable the detection of changes in enzyme activity independent of alterations in protein abundance, and provide a specific enrichment strategy for low-copy-number enzymes without interference from more abundant proteins. To date, ABPP has relied on gel-based methods for proteome analysis that are difficult to automate and often fail to resolve highly related protein species (5, 6, 9, 10). Here, we describe a gel-free platform for ABPP, termed Xsite, that permits the rapid and systematic quantification and identification of enzyme activities in complex protein mixtures. Materials and MethodsGeneral Materials and Methods. Fluorophosphonate probes were synthesized as described (5). Synthesis of the cathepsin probe, AX6429, is detailed in supplemental materials (Fig. 3, which is published as supporting information on the PNAS web site). Rat FAAH was expressed in Escherichia coli and purified as described (11). Porcine trypsin and equine butyrylcholinesterase were p...
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