Dye-sensitized photooxidation as a tool for the elucidation of critical amino acid residues in phosphoglucose isomerase.

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Rabbit muscle phosphoglucose isomerase was subjected to methylene blue and rose bengal sensitized photooxidation. This treatment caused the enzyme to lose activity quite rapidly with half‐lives of 2 to 10 minutes under the following conditions: pH 7.1; ionie strength, 0.11; temperature, 10°; enzyme concentration, 2 mg per ml. The time progress curve for inactivation was of apparent first order only until approximately 50% of the initial enzyme activity were lost. Quantitative rate studies were therefore restricted to a comparison of rates obtained during this initial period of the inactivation process. The thus obtained pseudo‐first rate constants for inactivation were dependent on reaction volume, enzyme concentration, pH, and ionic strenth.The loss in enzyme activity was accompanied by an initial decrease in the histidine, cysteine, and methionine content followed, after more prologned oxidation, by a decrease also in tryptophan and tyrosine. Other amino acid residues were not affected, as determined by monitoring the change in total amino acid composition simultaneously with the loss in enzyme activiy as a function of the photooxidation time.A direct correlation between the rate of activity loss and the oxidation rate of a specific amino acid residue could not be established because of the multiplicity of the photocatalytic modifications. Kinetic studies of the photoinactivation process, however, implicate histidine as the residue whose modification is primarily involved in the loss of catalytic activity. A direct participation of methionine is considered unlikely on the basis of the ionic strength dependency of photoinactivation, while cysteine has been excluded on the basis of other studies with organic mercurials.