The binding of Na+ to apo-enolase permits the binding of substrate

Enolase from rabbit muscle (PP-enolase) is inactivated by NaClO4. Enolase f ree of divalent cations is more susceptible to inactivation by NaClO4 than is enolase in the presence of Mg2+. We find that substrate protects apo-eno lase against inactivation, indicating that substrate can bind to enolase in the absence of a divalent cation. This binding is not due to contamination by trace levels of divalent cations since (1) it occurs even in the presen ce of EDTA or EGTA and (2) metal analysis by ICP (inductively coupled plasm a) mass spectrometry did not reveal sufficient contamination to account for the protection. The binding of PGA to apo-enolase did require Na+. When TM AClO(4) was used instead of NaClO4, there was no protection by PGA. Protect ion was restored when TMAClO(4) plus NaCl were used. The inactivation of ap o-enolase by NaClO4 is due to dissociation into inactive monomers. We concl ude that Na+ binds to apo-enolase, permitting substrate to then bind. Of th e three known Me2+ binding sites on enolase, we believe the most likely bin ding site for Na+ is the carboxylate cluster of site 1, the highest affinit y site of enolase. (C) 2000 Elsevier Science B.V. All rights reserved.