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  • Title: Effects of NAD+ binding on the luminescence of tryptophans 84 and 310 of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.
    Author: Gabellieri E, Rahuel-Clermont S, Branlant G, Strambini GB.
    Journal: Biochemistry; 1996 Sep 24; 35(38):12549-59. PubMed ID: 8823192.
    Abstract:
    The individual fluorescence and phosphorescence properties of W84 and W310 in Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase were identified through the construction of a single tryptophan mutant (W84F) and by comparison of the emission between mutant and wild-type enzymes. The results show that the luminescence of W310 is red-shifted and substantially quenched relative to that of W84. It displays an average subnanosecond fluorescence lifetime (tau F) and a very short, 50 microseconds, room-temperature phosphorescence (RTP) lifetime (tau P). The perturbation of W310 luminescence is believed to arise from a stacking interaction with Y283. In contrast, W84 exhibits a fluorescence lifetime tau F of several nanoseconds and a long-lived phosphorescence lifetime tau P, typical of buried, unperturbed TrP residues. NAD+ binding to the tetrameric enzyme causes a 55% reduction of W310 fluorescence intensity together with a nearly complete quenching of its low-temperature phosphorescence. W84, which is located far from the nicotinamide moiety of NAD+, is much less affected by the binding of the coenzyme; the reduction in fluorescence intensity is 35%, and its phosphorescence intensity is unchanged. Another consequence of NAD+ binding is a significant decrease of the RTP lifetime tau P of W84, manifesting thereby a conformational change in the region of the coenzyme-binding domain. However, no change is observed in the RTP lifetime tau P of W310 located in the catalytic domain. These findings and those obtained at partial coenzyme saturation support the conclusions derived from high-resolution crystallographic structures [Skarzynski, T., & Wonacott, A. J., (1988) J. Mol. Biol. 203, 1097-1118] that the NAD(+)-induced conformational change is sequential and that subtle rearrangement in the structure of unligated subunits might be responsible for the negative cooperative behavior of NAD+ binding.
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