The (βα)8-barrel enzyme indole-3-glycerol phosphate synthase (IGPS) catalyzes the multistep transformation of 1-(o- carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP) into indole-3-glycerol phosphate (IGP) in tryptophan biosynthesis. Mutagenesis data and crystal structure analysis of IGPS from Sulfolobus solfataricus (sIGPS) allowed for the formulation of a plausible chemical mechanism of the reaction, and molecular dynamics simulations suggested that flexibility of active site loops might be important for catalysis. Here we developed a method that uses extrinsic fluorophores attached to active site loops to connect the kinetic mechanism of sIGPS to structure and conformational motions. Specifically, we elucidated the kinetic mechanism of sIGPS and correlated individual steps in the mechanism to conformational motions of flexible loops. Pre-steady-state kinetic measurements of CdRP to IGP conversion monitoring changes in intrinsic tryptophan and IGP fluorescence provided a minimal three-step kinetic model in which fast substrate binding and chemical transformation are followed by slow product release. The role of sIGPS loop conformational motion during substrate binding and catalysis was examined via variants that were covalently labeled with fluorescent dyes at the N-terminal extension of the enzyme and mobile active site loop β1α1. Analysis of kinetic data monitoring dye fluorescence revealed a conformational change that follows substrate binding, suggesting an induced-fit-type binding mechanism for the substrate CdRP. Global fitting of all kinetic results obtained with wild-type sIGPS and the labeled variants was best accommodated by a four-step kinetic model. In this model, both the binding of CdRP and its on-enzyme conversion to IGP are accompanied by conformational transitions. The liberation of the product from the active site is the rate-limiting step of the overall reaction. Our results confirm the importance of flexible active loops for substrate binding and catalysis by sIGPS.