Role of arginine in the binding of thiamin pyrophosphate to Escherichia coli pyruvate oxidase.

The mode of interaction between Escherichia coli pyruvate oxidase and its cofactor, thiamin pyrophosphate (TPP), was studied with the aid of arginine-directed reagents. The enzyme is rapidly inactivated by either phenylglyoxal or 2,3-butanedione, with the cofactor, TPP, offering partial protection against these reagents. The inactivation by phenylglyoxal was found to be reversible. Experiments with [7-14C]phenylglyoxal showed that while several arginine residues react with this reagent, TPP can prevent the labeling of one such residue. Furthermore, inactivation by 2,3-butanedione is attended by at least a 100-fold decrease in affinity of the enzyme for TPP. These results suggest a direct role for arginine in the binding of the cofactor.