Ribose recognition by ribonuclease T1: difference spectral binding studies with guanosine and deoxyguanosine

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Abstract
The binding of RNase T1 [EC 3.1.4.8] with guanosine (Guo) and deoxyguanosine (dGuo) was studied in experiments employing UV difference spectroscopy in the pH range 3-9 at 0.2 M ionic strength and 25.degree. C. Similar experiments were conducted with .gamma.-carboxymethyl-glutamate-58 RNase T1 at pH 5.0. At most pH values the characteristic difference spectrum and association constant were obtained. The binding constant for dGuo was .apprx. 50 M-1 and did not significantly vary in the pH range 3.5-9.0. The binding constant for Guo increased from pH 3.5 to 5.0, was constant between pH 5.0 and 7.0 (.apprx. 3200 M-1) and decreased at higher pH values. The binding of Guo and dGuo with RNase T1 could also be distinguished in terms of the wavelength for maximal difference absorbance, .lambda.max, between pH 5.0 and 7.0. At higher and lower pH values, .lambda.max for Guo approached that found for dGuo. The value of the binding constant (.apprx. 6500 M-1) and the nature of the difference spectra for Guo and dGuo binding with .gamma.-carboxymethyl-glutamate-58-RNase T1 at pH 5.0 were identical. The discrete interaction of the Guo 2''-hydroxy group with RNase T1 probably involves the .gamma.-carboxylate of glutamate-58 and an imidazolium group at the active site.