Domain Bridging Interactions

Abstract

Aspartate transcarbamoylase undergoes a domain closure in the catalytic chains upon binding of the substrates that initiates the allosteric transition. Interdomain bridging interactions between Glu 50 and both Arg 167 and Arg 234 have been shown to be critical for stabilization of the R state. A hybrid version of the enzyme has been generated in vitro containing one wildtype catalytic subunit, one catalytic subunit in which Glu 50 in each catalytic chain has been replaced by Ala (E50A), and wild-type regulatory subunits. Thus, the hybrid enzyme has one catalytic subunit capable of domain closure and one catalytic subunit incapable of domain closure. The hybrid does not behave as a simple mixture of the constituent subunits; it exhibits lower catalytic activity and higher aspartate affinity than would be expected. As opposed to the wild-type enzyme, the hybrid is inhibited allosterically by CTP at saturating substrate concentrations. As opposed to the E50A holoenzyme, the hybrid is not allosterically activated by ATP at saturating substrate concentrations. Small angle x-ray scattering showed that three of the six interdomain bridging interactions in the hybrid is sufficient to cause the global structural change to the R state, establishing the critical nature of these interactions for the allosteric transition of aspartate transcarbamoylase.Escherichia coli aspartate transcarbamoylase catalyzes the committed step of pyrimidine biosynthesis, the condensation of carbamoyl phosphate and L-aspartate to form N-carbamoyl-Laspartate (1). The enzyme shows homotropic cooperativity for the substrate L-aspartate and is heterotropically regulated by ATP, CTP (1), and UTP in the presence of CTP (2). The holoenzyme is composed of six catalytic chains (M r 34,000), associated as two trimeric subunits, and six regulatory chains (M r 17,000), associated as three dimeric subunits. The active sites, three/ trimer, are located at the interface between catalytic chains, whereas the allosteric sites, two/dimer, are located on the Nterminal domain of the regulatory chains.Two functionally and structurally different states of aspartate transcarbamoylase have been characterized. The low affinity, low activity conformation of the enzyme is described as the "tense" or T state, whereas the high affinity, high activity conformation of the enzyme is described as the "relaxed" or R state. The conversion from the T state to the R state occurs upon aspartate binding to the enzyme in the presence of carbamoyl phosphate. Structurally, the enzyme elongates by at least 11 Å along the 3-fold axis, and the catalytic trimers and regulatory dimers rotate along their axes of symmetry, 10°and 15°, respectively (3). The allosteric transition can also be monitored in solution using small angle x-ray scattering (SAXS) 1 (4). In addition to the quaternary changes, several tertiary changes also occur because of a rearrangement of several important inter-and intrasubunit interactions. One important intrasubunit interaction that stabilizes the R state is between...

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