Role of allosteric: zinc interdomain region of the regulatory subunit in the allosteric regulation of aspartate transcarbamoylase from Escherichia coli.
The hydrophobic interface between the allosteric and the zinc domains of the regulatory subunit of aspartate transcarbamoylase has previously been implicated in the heterotropic ATP activation of the enzyme. The present work shows that this interface also affects CTP and CTP-UTP inhibition and proposes a structural explanation for the effects. Mutant enzymes derived from nonselective mutagenesis of residues r101-r106 (residues that contribute part of the interface) displayed a variety of homotropic and heterotropic effects. The cooperative behavior of the enzymes was affected, as indicated by reduced aspartate S0.5 values and apparent Hill coefficient values for V106L, V106L/N105S, and I103F/R102C. In addition, both ATP activation and CTP inhibition were significantly reduced and CTP+UTP synergistic inhibition was decreased in these mutants. The D104G mutant enzyme was subject to inhibition by CTP andCTP+UTP, but was not activated by ATP. Finally, the I103T mutant enzyme had an increased S0.5 value of 11.5 mM and displayed altered effector responses: ATP acted as an inhibitor, and the CTP+UTP synergistic inhibition was reduced. Most of these allosteric variations can be explained in terms of perturbations to the "tongue and groove" hydrophobic interface between the allosteric and the zinc domains and a consequent impact on a second interface ("reg1:cat4") between regulatory and catalytic subunits.
Rastogi
V K
VK
GEO-Centers, Inc./ERDEC, Building 3220, Aberdeen Proving Ground, Edgewood Area, Maryland, 21010, USA.
Swanson
R
R
Hartberg
Y M
YM
Wales
M E
ME
Wild
J R
JR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D011487:Protein Conformation; D015032:Zinc / Q000378:metabolism*
D003067:Coenzymes; D000255:Adenosine Triphosphate; D001221:Aspartate Carbamoyltransferase; D015032:Zinc