Coenzyme B12-dependent diol dehydrase: Chemical modification with 2,3-butanedione and phenylglyoxal

The apoenzyme of diol dehydrase was inactivated by two arginine-specific reagents, 2,3-butanedione and phenylglyoxal, in borate buffer. In both cases, the inactivation followed pseudo-first-order kinetics. Kinetic data show that the incorporation of a single reagent molecule per active site of the enzyme is necessary for the complete inactivation. The modification with 2,3-butanedione was reversed by dilution of the reagent and borate concentrations (65% activity recovered). 1,2-Propanediol (substrate) partially protected the enzyme against inactivation. The holoenzyme was almost insensitive to 2,3-butanedione and phenylglyoxal, indicating that the essential arginine residue is prevented from the attack of these reagents either by direct blockage with the bound coenzyme or by an indirect conformational change caused by coenzyme binding. The inactivation of diol dehydrase by 2,3-butanedione did not result in dissociation of the enzyme into subunits. From these results, we concluded that the essential arginine residue is located at or in close proximity to the active site of diol dehydrase.