Abstract
Abstract Dicarboxylic acid inhibitors and the amino and keto acid substrates interact with aspartate aminotransferase (EC 2.6.1.1) to form spectrally distinct enzyme-inhibitor and enzyme-substrate complexes. The spectra of solutions of the enzyme and varying concentrations of inhibitor or substrate are analyzed by computer methods to obtain the spectra of the enzyme-inhibitor and enzyme-substrate complexes. Programs have been written which permit the computation of the pKa values and the dissociation constants of the complexes with the use of the spectral data at many wave lengths. The complete spectra of the individual ionic species of the complexes are drawn. Comparison plots of the experimental spectral data and the calculated spectra can also be obtained. These computer methods are applied to four enzyme-inhibitor complexes and to complexes of the enzyme with its substrates and two pseudosubstrates. Spectra of apoaspartate aminotransferase containing bound pyridoxal phosphate analogues have also been analyzed.
Highlights
MethodsNlateriuZsThe aspartate aminotransferase was donated byDr W
These computer methods are applied to four enzyme-inhibitor complexes and to complexes of the enzyme with its substrates and two pseudosubstrates
The computer methods described here enable the calculation of pK, values, dissociation constants, and spectra of enzymeinhibitor and enzyme-substrate complexes
Summary
NlateriuZsThe aspartate aminotransferase was donated byDr W. T. Jenkins as the highly purified cytoplasmic a+subform [10]. The absorbance ratio AGo:A340was 3.6 in 0.02 M acetate buffer, pH 5.4. Enzyme concentrations were obtained from the absorbance at 364 rnp at pH 8.0, with the use of the molar extinction coefficient of 8.20 X lo3 [5, 11]. One mole is that amount of enzyme containing 1 mole of bound pyridoxal-P
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