Spectroscopic properties of the photoproducts of pyridoxal-5'-P irradiation: Catalytic site recognition of ribonuclease A.
Photoproducts of pyridoxal-5'-P, i.e., 4-pyridoxic-5'-P and bis-pyridoxal-5'-P, have been studied by spectroscopic methods. The spectroscopic properties of bis-pyridoxal-5'-P (bis-PLP) resemble those of pyridoxal-5'-P (PLP) under similar experimental conditions. The coupling of methylen hydrogens to the phosphorus atom has been shown by NMR spectroscopy. The singlet in the(31)P-NMR spectra and the triplet in(1)H-undecoupled experiments confirm the presence of the phosphate group in the 5' position of the structure of the vitamin. The effect of pH and solvent composition on the relative distribution of species of bis-pyridoxine-5'-P (bis-PNP) has been investigated by absorption and fluorescence spectroscopy. The acid-base dissociation of the phosphate group is easily detected by emission spectroscopy. Bis-PNP and bis-PLP bind to the enzyme RNase A and they behave as competitive inhibitors with respect to the substrate cytidine-2'-3'-cyclic phosphate. The natural forms of vitamin B6, pyridoxine, and pyridoxine-5'-P have no effect on the catalytic activity of the protein. Experimental evidence derived from fluorescence and inhibition experiments is consistent with the hypothesis that bis-PNP recognizes the catalytic site of RNase A.
Department of Biochemistry, University of Tennessee, 37916, Knoxville, Tennessee.
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