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Substrate deconstruction and the nonadditivity of enzyme recognition

Barelier S, Cummings JA, Rauwerdink AM, Hitchcock DS, Farelli JD, Almo SC, Raushel FM, Allen KN, Shoichet BK (2014) J Am Chem Soc 136, 7374-7382 PMCID: PMC4046767

The Computation Core spearheads the following investigation into the reactivity of enzymes toward substrate fragments. With input from the AH and HAD Bridging Projects, as well as enzymes produced and crystallized by the Protein and Structure Cores, researchers elucidate the ability of three enzymes classes to catalyze reactions with systematic fragments of known substrates. The results have broad implications for the utility of whole-metabolite versus metabolite-fragment screening libraries in the discovery of unknown substrates.

Abstract

Predicting substrates for enzymes of unknown function is a major postgenomic challenge. Substrate discovery, like inhibitor discovery, is constrained by our ability to explore chemotypes; it would be expanded by orders of magnitude if reactive sites could be probed with fragments rather than fully elaborated substrates, as is done for inhibitor discovery. To explore the feasibility of this approach, substrates of six enzymes from three different superfamilies were deconstructed into 41 overlapping fragments that were tested for activity or binding. Surprisingly, even those fragments containing the key reactive group had little activity, and most fragments did not bind measurably, until they captured most of the substrate features. Removing a single atom from a recognized substrate could often reduce catalytic recognition by 6 log-orders. To explore recognition at atomic resolution, the structures of three fragment complexes of the β-lactamase substrate cephalothin were determined by X-ray crystallography. Substrate discovery may be difficult to reduce to the fragment level, with implications for function discovery and for the tolerance of enzymes to metabolite promiscuity. Pragmatically, this study supports the development of libraries of fully elaborated metabolites as probes for enzyme function, which currently do not exist.

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Abstract Image:

Figure 1: Multiple ways of fragmenting the substrates of adenosine deaminase (ADA); MTA/SAH deaminase (SAHD); phosphoserine phosphatase (PSP); flavin mononucleotide phosphatase (FMP); and AmpC β-lactamase (AmpC). Only one fragment was typically tested at a time. For isoaspartyl dipeptidase (IAD), recognition of the component amino-acid monomers was investigated.

Figure 2: Stereoviews of key interactions within AmpC β-lactamase complexed structures. (A) The structure of AmpC in complex with 48 shows a covalent bond between Ser64 and the fragment substrate, (see also Figure S2 in the SI) and captures the stable acyl-enzyme intermediate step between the transition state acylation and deacylation complexes. (B) The structure of AmpC in complex with 45 shows the fragment in its product form, bound in two orientations to a distal subsite of the large overall binding site, interacting with Ser212, Tyr221, and Gly320.

Figure 3: Plot of % compound activity (as compared to the entire substrate) as a function of size. Size is measured as the number of heavy atoms.

Reprinted with permission from Barelier et al. J Am Chem Soc 136, 7374-7382. Copyright 2014 American Chemical Society.