Evidence that 31P NMR is a sensitive indicator of small conformational changes in the coenzyme of aspartate aminotransferase.
The pH dependence of 31P-NMR spectra of pig cytosolic aspartate aminotransferase, containing either N-(5'-phosphopyridoxyl)-L-aspartate or pyridoxal 5'-deoxymethylenephosphonate in place of the normal coenzyme pyridoxal 5'-phosphate, has been analysed. The chemical shifts of phosphopyridoxylaspartate and of pyridoxal 5'-deoxymethylenephosphonate model Schiff base in free solution show pK values of 6.3 and 7.4, attributable to the second deprotonation step of phosphate and phosphonate, respectively. However, these compounds behave very differently when bound to apoaspartate aminotransferase. 31P-NMR spectra of these enzyme derivatives indicate that the phosph(on)ate group remains dianionic throughout the pH range 4-8.5. A clear correlation between apparent pK values obtained from spectrophotometric titration of the coenzyme chromophore and those obtained by 31P NMR indicates that the same ionisation is being reported by both methods. The data are interpreted, on the basis of available crystallographic structures of chicken mitochondrial aspartate aminotransferase, to indicate that in each case the alteration in 31P chemical shift results from a conformational change in the coenzyme 5' side chain, in which one of the structures involves a near-eclipsed pair of bonds. Such a stressed conformation produces slight alterations in bond angles around the phosphorus atom, which in turn cause the observed change in 31P chemical shift. The evidence is taken to indicate that in this case 31P NMR is a sensitive reporter of stress in enzyme-bound pyridoxal 5'-phosphate and its derivatives.
Schnackerz
K D
KD
Physiologisch-Chemisches Institut der Universität Würzburg, FRG.
Wahler
G
G
Vincent
M G
MG
Jansonius
J N
JN
D000818:Animals; D001219:Aspartate Aminotransferases / Q000032:analysis*; D003067:Coenzymes / Q000032:analysis*; D003600:Cytosol / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008929:Mitochondria, Heart / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology*; D011487:Protein Conformation; D011732:Pyridoxal Phosphate; D013552:Swine
D003067:Coenzymes; D011732:Pyridoxal Phosphate; D001219:Aspartate Aminotransferases