During the reaction catalyzed by enolase, a mobile loop, residues 36-45, closes over the active site. In order to probe the role of this loop movement in catalysis, the glycines at positions 37 and 41 of rabbit muscle enolase (beta beta) have been mutated to alanines. The mutant forms-G37A and G41A-of enolase are both active, but have altered selectivity for divalent cations. G37A, when assayed with Mg(2+), has 12% the activity of the wild type. However, it is twice as active as wild type when assayed with Mn(2+), Zn(2+), or Co(2+). G41A has 4% the activity of the wild type with Mg(2+), is more active than wild type with Co(2+), and slightly less active than wild type with Mn(2+) and Zn(2+). The kinetic isotope effect for both mutants is greater than that of the wild type with all 4 divalent cations. These results indicate that the flexibility of this loop has subtle effects on catalytic activity.