JBC, Vol. 250, Issue 22, 8635-8641, Nov, 1975
Factors contributing to the inhibition of aspartate aminotransferase by dicarboxylic acids
S. M. Bonsib, R. C. Harruff and W. T. Jenkins
At pH 8.0 aspartate aminotransferase (L-aspartate:2-oxoglutarate
aminotransferase, EC 2.6.1.1) reacts with the modified substrate,
erythro-beta-hydroxy-L-aspartate, to form a mixture of enzyme-substrate
complexes absorbing at 492 nm. A variety of dicarboxylic acids were studied
spectrophotometrically as competitive inhibitors of this reaction. All of
the inhibitory dicarboxylic acids form a complex with the enzyme, absorbing
at 362 nm. In addition, some of the dicarboxylic acids form a protonated
complex absorbing at about 435 nm. This complex, which is the conjugate
acid of that absorbing at 362 nm, is formed only by those dicarboxylic
acids which can assume a configuration in which the two carboxyl groups are
positioned as in maleic acid. Bulky substituents, such as aromatic rings or
even methyl groups, prevent the formation of the protonated complex,
presumably because of steric restrictions at the active site. Substitution
of the central carbon atom of glutaric acid by heteroatoms of increasing
charge density results in a progressive decrease in inhibitory
effectiveness, at pH 8, primarily due to a loss of this pH-dependent
stabilization of the enzyme-dicarboxylic acid complex. Acids with an
aromatic ring are among the most potent dicarboxylic acid inhibitors of
this enzyme in spite of the fact that they do not undergo the pH-dependent
stabilization of their enzyme complexes. From these observations it was
concluded that the affinity of aspartate aminotransferase for dicarboxylic
acids is determined as much by the mechanism of binding as by the solvation
and steric effects.
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