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Biochemistry. 1998 Jun 16;37(24):8653-8.

The influence of the regulatory chain amino acids Glu-62 and IIe-12 on the heterotropic properties of Escherichia coli aspartate transcarbamoylase.

Author information

1
Boston College, Department of Chemistry, Merkert Chemistry Center, Chestnut Hill, Massachusetts 02167, USA.

Abstract

In the structure of Escherichia coli aspartate transcarbamoylase with CTP bound [Kosman, R. P., Gouaux, J. E., and Lipscomb, W. N. (1993) Proteins, Struct. Funct. Genet. 15, 147-177] Lys-6 and Glu-62 form a salt-link between two regulatory chains. However, recent X-ray structural studies suggest that side chain and backbone interactions existed between Glu-62 and Ile-12. Thus the interaction between Glu-62 and Ile-12 may help to establish the correct conformation of the nucleotide binding site. The present study of two single-site mutant enzymes, Glu-62r-->Ala and Ile-12r-->Ala, was undertaken to investigate whether the role of Glu-62 is to maintain the stability of the interface between the regulatory chains in the dimer, or interacts with the side chain of Ile-12r to define the nucleotide binding site. For both the mutant enzymes, the maximal velocity, the aspartate saturation at half the maximal velocity, and Hill coefficient were close to wild-type values. The Glu-62r-->Ala enzyme showed enhanced regulatory effects with ATP, CTP, and UTP. As a result of this mutation the enzyme losses its ability to discriminate between CTP and UTP. For the Ile-12r-->Ala enzyme, the heterotropic properties were reduced or eliminated. The enhanced regulatory effects observed with the Glu-62r-->Ala enzyme do not seem to be consistent with the presence of a salt-link between Glu-62r and Lys-6r. However, based upon kinetic data of the unique but completely opposite heterotropic properties of the two mutant enzymes, it is suggested that the side chain interaction between Glu-62r and Ile-12r helps to define the conformation of the effector binding pocket. In this study, we report the properties of both the Glu-62r-->Ala and Ile-12r-->Ala enzymes and their importance for the heterotropic activation and inhibition of aspartate transcarbamoylase.

PMID:
9628727
DOI:
10.1021/bi980456h
[Indexed for MEDLINE]

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