Genetic and Protein Engineering(10 Plenary Forums - 1 Event)

Biography

Biography: James A. Endrizzi

Abstract

Aspartate transcarbamoylase (ATCase) has been featured in many biochemistry textbooks as an example of allosteric enzyme regulation. It catalyzes the fi rst step of pyrimidine nucleotide biosynthesis and is feedback inhibited by cytidine triphosphate (CTP), which is the end product of the pathway. ATCase exhibits cooperative substrate binding to its catalytic subunits and allosteric binding of nucleotides to its regulatory subunits. Opposing inhibition by CTP and activation by adenosine triphosphate (ATP) promote homeostasis of purine and pyrimidine nucleotides. Regulation of ATCase activity has been interpreted as a ligand promoted change in the equilibrium between inactive (taut) and active (relaxed) states largely consistent with a two-state allosteric model. The structure of the ligand-free enzyme represents the inactive, taut state and the structure of ATCase bound to a bisubstrate analog (N-phosponacetyl-L-aspartate; PALA) has been proposed to represent the active, relaxed state. However, there is little evidence that the PALA-bound enzyme is structurally similar to the active conformation(s) in the absence of substrates. A novel approach to define the structure of the ligand free relaxed, or “activated” state is to use mutant enzymes that destabilize the taut state and shift the allosteric equilibrium toward the relaxed-state conformation(s). In contrast to the wild-type enzyme, which exhibits a more compact global conformation and sigmoidal enzyme kinetics, the mutants are more expanded and display hyperbolic kinetics characteristic of non-cooperative enzymes. Thus, we tested the hypothesis that ATCase is activated through the modulation of flexibility by determining the X-ray crystal structures of several activated-state mutants. We obtained crystals of three ATCase mutants in multiple crystal forms, collected X-ray diffraction data on the various forms and solved and refined dozens thus far. A variety of tertiary and quaternary structures in activated-state mutant enzymes support the hypothesis that these structures represent metastable states accessed by an ensemble of activated-state conformations..