VARIATIONS IN FLUORESCENCE AND ENZYMATIC-PROPERTIES OF BOVINE DIHYDROFOLATE REDUCTASE-CENTER-DOT-NADPH COMPLEX DURING THE SLOW CONFORMATIONAL CHANGE INDUCED BY COENZYME BINDING

When NADPH was added in large excess to bovine dihydrofolate reductase (H(2)folate reductase), there was a slow isomerization process betwee n two conformers of the binary complex (B-1 reversible arrow B-2), as shown by changes in the fluorescence properties. Thus, we monitored th e time dependence of (a) the quenching of protein intrinsic fluorescen ce intensity, (b) the polarization state of the fluorescence light emi tted by NADPH . H(2)folate reductase complexes and (c), from a more bi ological point of view, the enzymic activity of binary complex solutio ns. The kinetics for these three processes were in good agreement usin g the same temperature conditions. Furthermore, fluorescence studies p rovided information on the NADPH environment in the binary complex. As soon as NADPH bound to H(2)folate reductase, light emitted by the inv ariant Trp24 residue located within the coenzyme-binding site was quen ched by an energy-transfer process. Moreover, Trp57 and/or Trp113 emis sions were partially quenched. The subsequent NADPH-bound protein conf ormational change caused an additional quenching, probably of Trp57 an d/or Trp113 emissions. Thus, NADPH . H(2)folate reductase conformation was modified but no change was observed at the coenzyme-binding site, at least in our fluorescence study. These results were confirmed by p olarization measurements. The conformational change, as well as the in stantaneous NADPH binding, resulted in a more rigid form of the protei n, as shown by an increase in steady-state anisotropy values. Finally, the isomerization process led to a more active enzymic form.