EFFECTS OF NAD(-84 AND TRYPTOPHAN-310 OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM BACILLUS-STEAROTHERMOPHILUS() BINDING ON THE LUMINESCENCE OF TRYPTOPHAN)

The individual fluorescence and phosphorescence properties of W84 and W310 in Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydro genase were identified through the construction of a single tryptophan mutant (W84F) and by comparison of the emission between mutant and wi ld-type enzymes. The results show that the luminescence of W310 is red -shifted and substantially quenched relative to that of W84. It displa ys an average subnanosecond fluorescence lifetime (tau(F)) and a very short, 50 mu s, 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 fluorescen ce lifetime tau(F) Of several nanoseconds and a long-lived phosphoresc ence lifetime tau(p), typical of buried, unperturbed Trp residues. NAD (+) binding to the tetrameric enzyme causes a 55% reduction of W310 fl uorescence intensity together with a nearly complete quenching of its low-temperature phosphorescence. W84, which is located far from the ni cotinamide moiety of NAD(+), is much less affected by the binding of t he coenzyme; the reduction in fluorescence intensity is 35%, and its p hosphorescence intensity is unchanged. Another consequence of NAD(+) b inding is a significant decrease of the RTP lifetime tau(p) of W84, ma nifesting thereby a conformational change in the region of the coenzym e-binding domain. However, no change is observed in the RTP lifetime t au(p) Of W310 located in the catalytic domain. These findings and thos e obtained at partial coenzyme saturation support the conclusions deri ved 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 rearrang ement in the structure of unligated subunits might be responsible for the negative cooperative behavior of NAD(+) binding.