Intramolecular signal transmission in enterobacterial aspartate transcarbamylases - II. Engineering co-operativity and allosteric regulation in the aspartate transcarbamylase of Erwinia herbicola

The aspartate transcarbamylase (ATCase) from Erwinia herbicola differs from the other investigated enterobacterial ATCases by its absence of homotropi c co-operativity toward the substrate aspartate and its lack of response to ATP which is an allosteric effector (activator) of this family of enzymes. Nevertheless, the E. herbicola ATCase has the same quaternary structure, t wo trimers of catalytic chains with three dimers of regulatory chains ((c3) (2)(r2)(3)), as other enterobacterial ATCases and shows extensive primary s tructure conservation. In (c3)(2)(r2)(3) ATCases, the association of the catalytic subunits c(3) w ith the regulatory subunits r(2) is responsible for the establishment of po sitive co-operativity between catalytic sites for the binding of aspartate and it dictates the pattern of allosteric response toward nucleotide effect ers. Alignment of the primary sequence of the regulatory polypeptides from the E. herbicola and from the paradigmatic Escherichia coli ATCases reveals major blocks of divergence, corresponding to discrete structural elements in the E. coli enzyme. Chimeric ATCases were constructed by exchanging thes e blocks of divergent sequence between these two ATCases. It was found that the amino acid composition of the outermost beta-strand of a five-stranded beta-sheet in the effector-binding domain of the regulatory polypeptide is responsible for the lack of co-operativity and response to ATP of the E. h erbicola ATCase. A novel structural element involved in allosteric signal r ecognition and transmission in this family of ATCases was thus identified. (C) 1999 Academic Press.