NMR observation of exchangeable protons of pyridoxal phosphate and histidine residues in cytosolic aspartate aminotransferase.
Observation of the 93-kDa cytosolic aspartate aminotransferase by 500-MHz 1H NMR spectroscopy in H2O has revealed a series of resonances in the 10-18 ppm range arising from exchangeable protons. One of these (peak A) has been assigned to the proton bound to the ring nitrogen of the coenzyme pyridoxal 5'-phosphate. A second (peak B) is assigned to H143 which participates in a chain of hydrogen bonds that includes also the coenzyme-bound proton. There is a mutual nuclear Overhauser effect between these two resonances. Peaks A and B respond to changes in pH and to interaction of the enzyme with coenzyme derivatives and inhibitors. Peak A moves from 15.4 to 17.4 ppm as the pH is lowered, while peak B moves in the opposite direction from 14.7 to 13.7 ppm, both with an apparent pKa of 6.15. This pKa is associated with deprotonation of the imine nitrogen at the Schiff base linkage of the coenzyme with K258 of the enzyme. In spectra of enzyme containing pyridoxamine 5'-phosphate, peak A is observed at 16.5 ppm and peak B is at 13.9 ppm over a broad pH range. Peaks A and B are found at 17.8 and 14.0 ppm, respectively, for the enzyme complex with glutarate. When alpha-methylaspartate is added to the enzyme several new resonances appear in the spectrum, which are attributed to formation of the external aldimine. The position of peak A in spectra of various forms of the enzyme is interpreted to reflect the electronic distribution in the coenzyme ring. Several other peaks in this region of the spectrum also are sensitive to changes in pH or the addition of inhibitors. Some possible assignments of these resonances are discussed.
Kintanar
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A
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Metzler
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Metzler
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Scott
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RD
D001219:Aspartate Aminotransferases / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D011522:Protons; D011732:Pyridoxal Phosphate / Q000737:chemistry*
D011522:Protons; D006639:Histidine; D011732:Pyridoxal Phosphate; D001219:Aspartate Aminotransferases