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Protein Eng. 1994 Mar;7(3):413-24.

Characterization of the apparent negative co-operativity induced in Escherichia coli aspartate aminotransferase by the replacement of Asp222 with alanine. Evidence for an extremely slow conformational change.

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Department of Molecular and Cell Biology, University of California, Berkeley 94720.


The strictly conserved active site residue, Asp222, which forms a hydrogen-bonded salt bridge with the pyridine nitrogen atom of the pyridoxal 5' phosphate (PLP) co-factor of aspartate aminotransferase (AATase), was replaced with alanine (D222A) in the Escherichia coli enzyme. The D222A mutant exhibits non-hyberbolic saturation behavior with amino acid substrates which appear as apparent negative cooperativity in steady-state kinetic analyses. Single turnover progress curves for D222A are well described by the sum of two exponentials, contrasting with the monophasic kinetics of the wild-type enzyme. An active/inactive heterodimer containing the D222A mutation retains this biphasic kinetic response, proving that the observed cooperativity is not the result of induced allostery. The anomalous behavior is explained by a hysteretic kinetic model involving two slowly interconverting enzyme forms, only one of which is catalytically competent. The slow functional transition between the two forms has a half-life of approximately 10 mins. Preincubation of the mutant with the dicarboxylic inhibitor maleate shifts the equilibrium population of the enzyme towards the catalytically active form, suggesting that the slow transition is related to the domain closure known to occur upon association of this inhibitor with the wild-type enzyme. The importance of Asp222 in the chemical steps of transamination is confirmed by the approximately 10(5)-fold decrease in catalytic competence in the D222A mutant, and by the large primary C alpha-deuterium kinetic isotope effect (6.7 versus 2.2 for the wild-type). The transamination activity of the D222A mutant is enhanced 4- to 20-fold by reconstitution with the co-factor analog N-methylpyridoxal-5'-phosphate (N-MePLP), and the C alpha-proton abstraction step is less rate determining, as evidenced by the decrease in the primary kinetic isotope effect from 6.7 to 2.3. These results suggest that the conserved interaction between the protonated pyridine nitrogen of PLP and the negatively charged carboxylate of Asp222 is important not only for efficient C alpha-proton abstraction, but also for conformational transitions concomitant with the transamination process.

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