Threonine deaminase from Escherichia coli. II. Maturation and physical properties of the enzyme from a mutant altered in its regulation of gene expression.

D H Calhoun,J S Kuska,G W Hatfield

doi:10.1016/s0021-9258(19)41990-x

Abstract

The biosynthetic L-threonine deaminase (L-threonine hydrolase deaminating, EC 4.2.1.16) has been purified from Escherichia coli K12 regulatory mutant CU18. This mutant has properties that follow the predictions of the autogregulatory model previously proposed for the control of synthesis of the isoleucine-valine biosynthetic enzymes. The autoregulatory model specifies that L-threonine deaminase participates in the control of the expression of the ilv ADE gene cluster as well as the ilv B gene and ilv C gene, which constitute three separate units of regulation. The single mutation in strain CU18 results in altered regulation of ilv gene expression and in the production of an altered L-threonine deaminase. The immature form of the enzyme purified from mutant CU18 exhibits an altered response to L-valine, a maturation-inducing ligand. The native form of the mutant is altered in its apparent Km for L-threonine and in its response to the effects of L-valine and L-isoleucine upon catalytic activity. The mutant and wild type L-threonine deaminases differ in the apoenzyme formed as a consequence of alkaline dialysis. Dialysis of the mutant enzyme yields an apoenzyme mixture, apparently of dimers and monomers, while the wild type enzyme yields only dimers. The CU18 L-threonine deaminase, is however, indistinguishable from the wild type enzyme in molecular weight and subunit composition.

Highlights

In this report we demonstrate that the L-threonine deaminase purified from mutant CU18 is altered in several of its kinetic and physical properties
What has not been understood, is: (a) the nature of the cellular mechanisms employed in the repression process to monitor the levels of and requirement for the branched chain amino acids, and (b) the molecular events involved in translating this signal of excess end products into a specific act&; namely, the reduced rate of synthesis of the ilv gene products
The native form of the mutant enzyme is altered in its apparent K, for L-threonine and in its response to the effects of r.-valine and r,-isoleucine upon catalytic activity

Summary

Methods

Enzyme Purijication-The methods used for purification of the enzyme, sedimentation equilibrium experiments, and sodium dodecyl sulfate gel electrophoresis have been previously described [9]. The n-isoleucine concentration in all buffers used in the enzyme purification procedure [9] was increased lo-fold to 1 X 1OW M to stabilize the mutant n-threonine deaminase. In order to increase the yield of enzyme during the purification procedure, we employed an additional step with both the wild type and mutant enzymes. The final enzyme preparation from the previously described procedure [9] was diluted lo-fold into buffer containing (per liter) : 1 X 10-I mol of potassium phosphate buffer, pH 6.25, 1 X 10-s mol of EDTA, 1 X 10m2 mol of L-isoleutine, 5 X lo-” mol of dithiothreitol,.and

Results

Discussion

Conclusion