Proton magnetic resonance studies of glutamate-alanine transaminase-catalyzed deuterium exchange. Evidence for proton conservation during prototropic transfer from the alpha carbon of L-alanine to the C4-position of pyridoxal 5'-phosphate.
Pulsed Fourier transform proton magnetic resonance spectroscopy was used to study the glutamate-alanine transaminase-catalyzed incorporation of deuterium from solvent deuterium oxide into the alpha and beta positions of L-alanine. It was found that the beta proton resonance signal initially disappears slightly faster than the signal due to the alpha proton, but whereas the alpha proton signal decays exponentially, that due to the beta proton signal does not. Eventually, the rate of decrease of the alpha proton signal becomes greater than that for the beta proton. This change in the relative rates is ascribed to a deuterium isotope effect upon substitution of an alpha proton by a deuteron. Furthermore, as deuterium begins to replace hydrogen, two classes of alanine become distinguishable, i.e. alanine which contains deuterium in the alpha position and hydrogen in the beta position, and alanine which contains hydrogen in the alpha position and deuterium in the beta position. Thus, removal of all 3 beta protons is not contingent upon loss of an alpha proton from the same molecule. The two classes of deuterated alanine may conceivably arise by a scrambling mechanism in which protons are transferred from the alpha to the beta position and vice versa. Present evidence excludes this scramblong mechanism and leads to the conclusion that deuterium incorporation into L-alanine involves, (a) the reversible enzymatic conversion of the classical ketimine enzymes intermediate to an enaminetype structure, and (b) considerable conservation of label during the prototropic shift from the alpha carbon of L-alanine to the C4-position of pyridoxal 5'-phosphate. It is also postulated that alanine binds at the active site in such a way as to bring the beta protons into close contact with a basic group on the enzyme surface. This group is distinct from that used in abstraction of an alpha proton. The beta protons of glutamate are not enzymatically removed; presumably glutamate binds in such a way that the beta protons cannot effectively interact with an enzyme base. Similar studies were carried out on soluble glutamate-aspartate transaminase; no evidence was found for significant enzyme-catalyzed deuterium incorporation into the beta position of L-glutamate, L-aspartate, and L-alanine.
D000409:Alanine; D000410:Alanine Transaminase / Q000378:metabolism; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D003903:Deuterium; D005583:Fourier Analysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D011487:Protein Conformation; D011732:Pyridoxal Phosphate; D013696:Temperature
D011732:Pyridoxal Phosphate; D003903:Deuterium; D000410:Alanine Transaminase; D000409:Alanine