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J. Biochem, 1970, Vol. 68, No. 1 69-79
© 1970 Japanese Biochemical Society

research-article

Studies on the Photooxidation of Ribonuclease T1

Masachika IRIE

Faculty of Pharmaceutical Sciences, Kyoto University Kyoto

1. Photooxidation of ribonuclease T1(EC 2.7.7.26 [EC] ) was studied to obtain information on the active center of the enzyme. The effect of pH on the photo-inactivation rate indicated the involvement of a group having a pKa value of 7.5 in the activity. This value is coincident with that for one of the functional groups which have been detected from kinetic studies.

2. Amino acid analysis of photooxidized RNase T1, possessing only 2–3% of the original activity, showed that two histidine residues had been destroyed leaving the tryptophan residue mostly intact.

3. Photooxidation in the presence of 2‘, (3’)-GMP resulted in the oxidation of only one histidine residue without any loss of the enzyme activity.

4. Alkaline titration of tyrosine residues, fluorescence emission spectrum, and difference spectrum occuring on exposure of the photooxidized enzyme to 6 M guanidine hydrochloride all indicated that photooxidation does not significantly affect the gross, conformation around the tyrosine and tryptophan residues of the enzyme.

5. The photooxidized enzyme could also combine with 2‘, (3’)-GMP, though the affinity was about 4-fold lower than that for the native enzyme and was similar to that for the carboxymethylated enzyme.

6. The photooxidized enzyme could bind a substrate analog, guanylyl(2‘{uparrow}5’)guanosine, as evidenced by the nucleotide binding by the native enzyme. This suggested that oxidized enzyme could also interact with the substrate guanylly(3‘{uparrow}5’) guanosine.

7. The difference spectra caused by the binding of the inhibitor and substrate analog nucleotides to the photooxidized enzyme were qualitatively similar to those observed with the native enzyme, suggesting that the two histidine residues sensitive to photooxidation are not involved in the interaction of the base moieties of these nucleotides with the enzyme protein.


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