THE EFFECTS OF SODIUM-PERCHLORATE ON RABBIT MUSCLE ENOLASE - SPECTRALCHARACTERIZATION OF THE MONOMER

Incubation of rabbit beta beta enolase in NaClO4 (less than or equal t o 0.3 M) results in a loss of enzymatic activity and striking changes in the second-derivative ultraviolet spectrum of enolase. HPLC gel fil tration shows that dissociation of the dimeric enzyme is occurring. We have used molecular modelling, fluorescence and circular dichroic spe ctroscopy to examine the structural differences between the monomeric and dimeric forms of this protein. In the dimer, the tyrosine residues are in a non-polar environment; upon dissociation, two of them that w ere at the dimer interface become exposed. This results in large chang es in the second-derivative spectrum. Both the tryptophan fluorescence emission spectrum and the aromatic region of the CD spectrum indicate that there are also changes in the environment of other aromatic resi dues. No perturbations in the peptide bond region of the CD spectrum a re observed. We propose that the major structural effect of NaClO4 is to increase the flexibility of the loops connecting the helices and st rands of the alpha/beta barrel of enolase. These loops, which contain about half of the aromatic residues, contain some of the residues of t he active site and other residues involved in subunit contacts, Increa sed flexibility of the loops could disrupt both subunit interactions a nd the structure of the active site.