Linkage between catalytic mechanism and conformational dynamics in (beta/alpha)8-barrel enzymes
Biophysics
Structural Biology
Final Report Abstract
Enzymes perform reactions with remarkable catalytic efficiency, selectivity and specificity and their function is closely linked to their molecular motions. Along these lines, the process of catalysis typically involves movements of residues in the active site of the enzyme during substrate binding and product release. These steps include motions of single residues as well as opening and closing of loop regions or entire domains. Such ligand-driven conformational changes are very well documented and are grouped under the umbrella term “induced-fit motions”, stating that binding of the substrate leads to the transition of non-productive and often poorly defined active site conformations into a single well-defined conformation that is complementary to the reaction transition state. Nevertheless, the direct role of enzyme motions in accelerating the individual states of the catalytic reaction is still under debate. We have studied this problem on the example of the mobile active site β1α1-loop (loop1) of the (βα)8-barrel enzyme HisF, which is the cyclase subunit of imidazole glycerol phosphate synthase. Loop1 variants containing single mutations of conserved amino acids showed drastically reduced rates for the turnover of the substrates N´-[(5´-phosphoribulosyl) formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PrFAR) and ammonia to the products imidazole glycerol phosphate (ImGP) and 5-aminoimidazole-4-carboxamide-ribotide (AICAR). A comprehensive mechanistic analysis including stopped-flow kinetics, X-ray crystallography, NMR spectroscopy, and MD simulations detected three conformations of loop1 (open, detached, closed) whose populations differed between wild-type HisF and functionally affected loop1 variants. Transient stopped-flow kinetic experiments demonstrated that wt-HisF binds PrFAR by an induced-fit mechanism whereas catalytically impaired loop1 variants bind PrFAR by a simple two-state mechanism. Our findings suggest that PrFAR-induced formation of the closed conformation of loop1 brings active site residues in a productive orientation for chemical turnover, which we show to be the rate-limiting step of HisF catalysis. After the cyclase reaction, the closed loop conformation is destabilized, which favors the formation of detached and open conformations and hence facilitates the release of the products ImGP and AICAR. Our data demonstrate how different conformations of active site loops contribute to different catalytic steps, a finding that is presumably of broad relevance for the reaction mechanisms of (βα)8-barrel enzymes and beyond.
Publications
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Conformational Modulation of a Mobile Loop Controls Catalysis in the (βα) 8 -Barrel Enzyme of Histidine Biosynthesis HisF. JACS Au, 4(8), 3258-3276.
Hupfeld, Enrico; Schlee, Sandra; Wurm, Jan Philip; Rajendran, Chitra; Yehorova, Dariia; Vos, Eva; Ravindra, Raju Dinesh; Kamerlin, Shina Caroline Lynn; Sprangers, Remco & Sterner, Reinhard
