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chemistry. Chirality, 19: 589–600. E-mail: [email protected]ac.jp Small Structural Change Producing Tryptophanase Activity on D-tryptophan Akihiko Shimada Sustainable Environmental LY294002 Studies, Graduate School of Life and Environmental Sciences, University
of Tsukuba, Tsukuba, Japan Tryptophanase (TPase) is an enzyme with extremely tight stereospecificity, cleaving l -tryptophan into indole, having no activity on D-tryptophan under ordinary conditions. However, it becomes active toward d-tryptophan in highly concentrated ammonium phosphate solutions quite different from what was expected. The only salts inducing the reaction were diammonium selleck compound phosphate, triammonium phosphate and ammonium sulfate, although other salts didn’t have the activity at all. Free tryptophan is more readily influenced by alkaline pH or strong ion strengths than other biological amino acids. If ammonium phosphates affect chemical racemization on D-tryptophan, the enzymological significance of this reaction is lost. So it is important to demonstrate that ammonium phosphates do not racemize free D-tryptophan at all. We used an HPLC column appropriate for tryptophan resolution to analyze free D-tryptophan, demonstrating that the reaction is enzymatic metabolism (Shimada, 2007). Ammonium phosphates as diammonium Crenigacestat order hydrogenphosphate or triammonium phosphate probably produce
structural change in tryptophanase, which makes it possible that activity on D-tryptophan will emerge. This result indicates enzyme stereospecificity Terminal deoxynucleotidyl transferase is more flexible than we think. Judging from the flexibility of tryptophanase stereospecificity, this conformational change is maybe small. Circular dichroism analyses were thus applied to tryptophanase in ammonium phosphate solution. A 200 μL of monoammonium hydrogenphosphate (MAP), diammonium hydrogenphosphate (DAP), and triammonium phosphate (TAP) of 50% saturation and phosphate buffer (PB) solutions with 0.5 μM of apoTPase and 1.1 mM of PLP was injected in a 0.1 cm path length cell in a circular dichroism (CD) spectrophotometer. Spectra were recorded at wavelengths from 200 to 350 nm at room temperature. Five scans were repeated per a spectrum, averaged, and expressed as molar ellipticity in degrees cm2 dmol -1.