The contribution of individual interchain interactions to the stabilization of the T and R states of Escherichia coli aspartate transcarbamoylase.
Stabilization of the T and R allosteric states of Escherichia coli aspartate transcarbamoylase is governed by specific intra- and interchain interactions. The six interchain interactions between Glu-239 in one catalytic chain of one catalytic trimer with both Lys-164 and Tyr-165 of a different catalytic chain in the other catalytic trimer have been shown to be involved in the stabilization of the T state. In this study a series of hybrid versions of aspartate transcarbamoylase was studied to determine the minimum number of these Glu-239 interactions necessary to maintain homotropic cooperativity and the T allosteric state. Hybrids with zero, one, and two Glu-239 stabilizing interactions do not exhibit cooperativity, whereas the hybrids with three or more Glu-239 stabilizing interactions exhibit cooperativity. The hybrid enzymes with one or more of the Glu-239 stabilizing interactions also exhibit heterotropic interactions. Two hybrids with three Glu-239 stabilizing interactions, in different geometric relationships, had identical properties. From this and previous studies, it is concluded that the 239 stabilizing interactions play a critical role in the manifestation of homotropic cooperativity in aspartate transcarbamoylase by the stabilization of the T state of the enzyme. As substrate binding energy is utilized, more and more of the T state stabilizing interactions are relaxed, and finally the enzyme shifts to the R state. In the case of the Glu-239 stabilizing interactions more than three of the interactions must be broken before the enzyme shifts to the R state. The interactions between the catalytic and regulatory chains and between the two catalytic trimers of aspartate transcarbamoylase provide a global set of interlocking interactions that stabilize the T and R states of the enzyme. The substrate-induced local conformational changes observed in the structure of the isolated catalytic subunit drive the quaternary T to R transition of aspartate transcarbamoylase and functionally induced homotropic cooperativity.
Sakash
J B
JB
Department of Chemistry, Boston College, Merkert Chemistry Center, Chestnut Hill, Massachusetts 02467, USA.
Kantrowitz
E R
ER
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D020134:Catalytic Domain; D003570:Cytidine Triphosphate / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D020035:Holoenzymes / Q000737:chemistry; D007700:Kinetics; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology
D020035:Holoenzymes; D001224:Aspartic Acid; D003570:Cytidine Triphosphate; C013195:sparfosic acid; D000255:Adenosine Triphosphate; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid