VARIATIONS IN FLUORESCENCE AND ENZYMATIC-PROPERTIES OF BOVINE DIHYDROFOLATE REDUCTASE-CENTER-DOT-NADPH COMPLEX DURING THE SLOW CONFORMATIONAL CHANGE INDUCED BY COENZYME BINDING
O. Rimet et al., VARIATIONS IN FLUORESCENCE AND ENZYMATIC-PROPERTIES OF BOVINE DIHYDROFOLATE REDUCTASE-CENTER-DOT-NADPH COMPLEX DURING THE SLOW CONFORMATIONAL CHANGE INDUCED BY COENZYME BINDING, European journal of biochemistry, 228(1), 1995, pp. 55-59
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.