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The complex of ribonuclease A (RNase A) with uridine vanadate (U-V), a
transition-state analogue, has been studied with 51V and proton NMR spectroscopy
in solution and by neutron diffraction in the crystalline state. Upon the
addition of aliquots of U-V at pH 6.6, the C epsilon-H resonances of the two
active-site histidine residues 119 and 12 decrease in intensity while four new
resonances appear. Above pH 8 and below pH 5, these four resonances decrease in
intensity as the complex dissociates. These four resonances are assigned to
His-119 and His-12 in protonated and unprotonated forms in the RNase-U-V
complex. These resonances do not titrate or change in relative area in the pH
range 5-8, indicating a slow protonation process, and the extent of protonation
remains constant with ca. 58% of His-12 and ca. 26% of His-119 being protonated.
The results of diffraction studies show that both His-12 and His-119 occupy
well-defined positions in the RNase-U-V complex and that both are protonated.
However, while the classic interpretation of the mechanism of action of RNase
based on the proposal of Findlay et al. [Findlay, D., Herries, D. G., Mathias,
A. P., Rabin, B. R., & Ross, C. A. (1962) Biochem. J. 85, 152-153] requires
both His-12 and His-119 to be in axial positions relative to the pentacoordinate
transition state, in the diffraction structure His-12 is found to be in an
equatorial position, while Lys-41 is close to an axial position. Hydrogen
exchange data show that the mobility and accessibility of amides in the
RNase-U-V complex do not significantly differ from what was observed in the
native enzyme. The results of both proton NMR in solution and neutron
diffraction in the crystal are compared and interpreted in terms of the
mechanism of action of RNase.
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