Proton Magnetic Resonance Studies of Glutamate-Alanine Transaminase-catalyzed Deuterium Exchange EVIDENCE FOR PROTON CONSERVATION DURING PROTOTROPIC TRANSFER FROM THE a CARBON OF L-ALANINE TO THE C’,-POSITION OF PYRIDOXAL 5’-PHOSPHATE*

Pulsed Fourier transform proton magnetic resonance spectroscopy was used to study the glutamatealanine transaminase-catalyzed incorporation of deuterium from solvent deuterium oxide into the cy and p positions of L-alanine. It was found that the /3 proton resonance signal initially disappears slightly faster than the signal due to the LY proton, but whereas the o( proton signal decays exponentially, that due to the p proton signal does not. Eventually, the rate of decrease of the 01 proton signal becomes greater than that for the fl proton. This change in the relative rates is ascribed to a deuterium isotope effect upon substitution of an (Y 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 cy position and hydrogen in the fl position, and alanine which contains hydrogen in the LY position and deuterium in the p position. Thus, removal of all 3 p protons is not contingent upon loss of an LY 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 cy to the p position and vice uersa. Present evidence excludes this scrambling mechanism and leads to the conclusion that deuterium incorporation into L-alanine involves, (a) the reversible enzymatic conversion of the classical ketimine enzyme intermediate to an enaminetype structure, and (b) considerable conservation of label during the prototropic shift from the LY carbon of L-alanine to the C’,-position of pyridoxal 5’-phosphate. It is also postulated that alanine binds at the active site in such a way as to bring the fi protons into close contact with a basic group on the enzyme surface. This group is distinct from that used in abstraction of an o( proton. The /3 protons of glutamate are not enzymatically removed; presumably glutamate binds in such a way that the fi protons cannot effectively interact with an enzyme base. Similar studies were carried out on soluble glutamate-aspartate transaminase; no evidence was fo;nd for significant enzyme-catalyzed deuterium incorporation into the p position of L-glutamate, L-aspartate, and L-alanine.