Regulation Reversal Mutation: Characterization of End Product-activated Mutants of Bacillus subtilis

Abstract

Abstract The physical and enzymatic properties of prephenate dehydratase from three regulatory mutants and the wild type of Bacillus subtilis are compared. The mutants were selected on the basis of their resistance to growth inhibition by the phenylalanine analogue β-thienylalanine. The three mutant enzymes either are resistant to feedback inhibition and are inherently more active than the enzyme from the parent strain or are capable of being activated by l-phenylalanine, the feedback inhibitor of the wild type enzyme. We have termed the gene modification giving rise to these end product-activated mutants regulation reversal mutation. The wild type enzyme is protected from heat and storage inactivation by its inhibitor, phenylalanine. In the presence of phenylalanine the enzyme gives substrate saturation curves which are not strictly first order. The Km for barium prephenate is ≅5 x 10-3 m. A molecular weight of approximately 65,000 was determined by sucrose gradient centrifugation. The prephenate dehydratase from mutant WB740 is resistant to phenylalanine inhibition, is protected from heat denaturation by the substrate barium prephenate but not by phenylalanine, and shows Michaelis-Menten kinetics in substrate saturation curves. The Km for barium prephenate is 8.3 x 10-4 m. The molecular weight is approximately 190,000, roughly 3 times the value obtained for the wild type enzyme. A second thienylalanine-resistant mutant, WB2136, has two readily separable species of prephenate dehydratase which differ from each other in several characteristics. One species resembles the enzyme from WB740 in molecular weight (approximately 190,000) and heat stability characteristics. However, unlike WB740, it is extremely labile when stored at 0° in the absence of phenylalanine. Substrate saturation curves reveal Michaelis-Menten kinetics. The Km for barium prephenate is 2.3 x 10-3 m. The second species resembles the wild type enzyme in molecular weight (approximately 65,000) and heat stability characteristics. This species gives sigmoid substrate saturation curves. Although interconversion of the two enzymes could not be demonstrated in vitro, conversion in vivo very likely occurs, since the mutation to end product activation is expressed in both enzyme species. A third thienylalanine-resistant mutant, WB2155, has a single species of prephenate dehydratase, which is capable of being activated by phenylalanine. The molecular weight of this enzyme is approximately 65,000, the same as the wild type enzyme and the light enzyme of WB2136. Substrate saturation curves (determined in the presence of the activator) were nearly identical with those observed for the light enzyme of WB2136. The enzyme is not protected from heat inactivation by either phenylalanine or barium prephenate. The sites concerned with analogue resistance in all three mutants map within, or extremely close to, the structural gene for prephenate dehydratase.