Abstract
Resonance Raman (RR) spectra are reported for aspartate aminotransferase from pig heart cytosol, and for inhibitor complexes. They are interpreted with reference to the previously analyzed spectra of pyridoxal phosphate (PLP) Schiff base adducts. This comparison shows that, as expected, the pyridine N atom is protonated in the native enzyme at pH 5, and in the glutarate complexes at pH 8.5, and that it is also protonated in the alpha-methylaspartate complex; the stabilization of the pyridine proton at high pH must be due to the interaction with aspartate 222 seen in the x-ray crystal structure. RR spectra of the erythro-beta-hydroxy-DL-aspartate complex, representing the p-quinoid enzyme intermediate, as well as of AlIII complexes of PLP Schiff bases with phenylalanine and tyrosine ethyl ester have been obtained via the coherent anti-Stokes Raman scattering technique, and partially assigned. A novel H/D exchange at the coenzyme C4' atom has been observed for the native enzyme in D2O, and has been determined, by a combination of NMR and RR measurements, to be due to the Raman laser irradiation. This photoprocess, which is not observed for PLP Schiff bases in aqueous solution, is attributed to a photoexcited p-quinoid intermediate, similar to that implicated in the enzyme mechanism. It is suggested that this intermediate is stabilized by protein interactions which localize charge on the phenolate O atom, plausibly a hydrogen bond from the nearby tyrosine 225. H/D exchange would then follow via the aldimine-ketimine interconversion known to take place in the enzyme reaction.
Highlights
Resonance Raman (RR) spectra a r e reported for as- from aspartate to ketoglutarate, or the reverse reaction
RR spectra of theerythro-8-hydroxy-DL-aspartate complex, representing the p-quinoid enzyme intermediate, as well as of Al"' complexes of PLP Schiff bases with phenylalanine and tyrosine ethyl ester have been obtained via the coherent antiStokes Raman scattering technique, and partially assigned
The spectra establish that the pyridine N atom of the coenzyme is protonated in allthese states and make it likely that the pyridinum group remains H-bonded to a protein residue throughout the enzymatic reaction
Summary
EVIDENCE FOR PYRIDINEPROTONATION AND A NOVEL PHOTOCHEMICAL H/D EXCHANGE A T THE IMINE CARBONATOM*. For the quinoid adduct of AAT, formed with erythro-/3-hydroxy-DL-aspartatew,e found interference from fluorescence to be too intense for spontaneous RR spectroscopy, and utilized the coherent anti-Stokes Raman scattering technique (11)to record the RR spectrum, comparing it to similar spectra of model complexes between Al"' and pyridoxal Schiff bases of phenylalanine and tyrosine ethyl esters. Glutarate binds to the AAT active site without disrupting the PLP Schiff base linkage to the side chain of lysine 258, the "internal" aldimine This complex has positive circular berlite CG-50 column in the NH,+ form, and eluted with 20 mM dichroism associated with its 430-nm absorption band (4), ammonium acetate buffer, pH 4.7. NMR showed 94% replacement of generated by disymmetric interactions of the chromophore the 4'-H by 'H
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