Glutamic-Aspartic Transaminase
IX. EQUILIBRIA WITH GLUTAMATE AND 
-KETOGLUTARATE
W. Terry Jenkins 1 and Linda D'Ari 1
From the
1 From the Department of Biochemistry, University of California, Berkeley, California 94720
A detailed analysis is presented of the spectral changes produced in solutions of aminotransferases under the following experimental conditions: (a) the keto acid concentration is held constant; (b) the keto acid concentration; is made to be proportional to the free amino acid concentration; (c) no keto acid is added; (d) in places of keto acid, another amino acid substrate or substrate analogue is added which will react with either the phosphopyridoxal or phosphopyridoxamine form of the enzyme to form a complex absorbing at a unique wave length where neither free form absorbs.
When these methods were used to determine the dissociation constants for glutamate and ketoglutarate complexes with the isomer of the pig heart extramitochondrial glutamic-aspartic transaminase (EC 2.6.1.1), consistent results were obtained.
erythro-ß-Hydroxy-l-aspartate was shown to have a high affinity for the phosphopyridoxamine form of this enzyme. This behavior is unique, for neither l-aspartate nor l-glutamate reacts similarly.
The phosphopyridoxamine form of the enzyme could be prepared, free of bound substrate, by dialysis first against radioactive glutamate and then against buffer.
The spectra of enzyme-substrate complexes were compared for four different substrates. It is suggested that at least four species are indicated. If there are several species, the equilibria between them are not pH-sensitive.
All of the results which were obtained were consistent with the pyridoxal-pyridoxamine enzyme hypothesis.
Submitted on December 23, 1965
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