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.
Using cloned DNA probes specific for two isoforms of cardiac myosin light chains (MLCs), nonphosphorylatable MLC1 and phosphorylatable, regulatory MLC2, we have observed that the MLC1 messenger RNA of ventricular type does not appear in detectable amounts in atrial cells of either normotensive Wistar-Kyoto rat strain (WKY) or spontaneously hypertensive rat strain (SHR). The messenger RNA of regulatory isoform of ventricular MLC2, on the other hand, is found in threefold excess in atria of SHR relative to that of age-matched WKY. The increased level of MLC2 messenger RNA is present even in 6-week-old SHR atria where there is no established overloading of the heart. Thus, it appears that the increased expression of the regulatory MLC2 gene in SHR atrial cells is a predetermined event, which, most likely, participates in functional adaptation of the myocardium in response to pressure overload and subsequent hypertrophy.
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