Picosecond laser fluorometry of FAD of D-amino acid oxidase-benzoate complex

Abstract

Formation of a complex of D-amino acid oxidase (D-amino acid:O2 oxidoreductase (deaminating), EC 1.4.3.3) and benzoate, an enzyme-substrate complex model, was studied by measuring the fluorescence life-time of the coenzyme FAD of the complex by using a mode-locked Nd:YAG laser and a streak camera. The value of lifetime was 60 +/- 10 ps in the monomer of the complex and it was extremely short (much less than 5 ps) in the dimer of the complex. Since the values of fluorescence lifetime of the coenzyme are 130 ps in the monomeric form of free enzyme and 40 ps in the dimeric form of free enzyme, the decrease in the lifetime upon complex formation with benzoate is slight in the monomer (reduced to one-half) whereas marked in the dimer (reduced to less than 1/10). By analyzing the fluorescence decay curve, a dissociation constant of the monomer-dimer equilibrium of the complex was evaluated to be 0.4 +/- 0.3 microM, which is much smaller than that in free enzyme. Fluorescence analysis under steady state excitation revealed that the apparent dissociation constant (K) of FAD from the enzyme was decreased by 1:1000 upon the complex formation. Relative quantum yield of the fluorescence of FAD in the complex to that of free FAD exhibited appreciable dependence on the complex concentration: greater in the monomer and less in the dimer. These results suggest that a molecular interaction between FAD and amino acid residue(s) is strengthened by the complex formation, which contributes to a remarkable conformational change in the protein moiety of the complex.