Discrimination between nucleotide effector responses of aspartate transcarbamoylase due to a single site substitution in the allosteric binding site.
The substitution of alanine for lysine at position 56 of the regulatory polypeptide of aspartate transcarbamoylase affected both homotropic and heterotropic characteristics. In the absence of effectors, the ALAr56-substituted holoenzyme lost the homotropic cooperativity observed for aspartate in the wild-type holoenzyme. Under conditions of allosteric inhibition in the presence of 2mM CTP, the cooperative character of ATCase was restored, and the Hill coefficient increased from 1.0 to 1.7. In contrast to the native enzyme, the altered enzyme did not respond to ATP; however, ATP could still bind to the enzyme as demonstrated by its direct competition with CTP. Furthermore, the recently observed CTP-UTP synergism of the wild-type enzyme was not detectable. The site-directed mutant enzyme could not be activated by low levels of the bisubstrate analogue, N-(phosphonacetyl)-L-aspartate, and the rate of association of pHMB with the cysteine residues located at the interface of the catalytic and regulatory chains was slightly altered. These characteristics suggested that the mutant holoenzyme assumed a relaxed (or abnormal T state) conformation. Thus, this single substitution differentially affected the heterotropic responses to the various allosteric effectors of ATCase and eliminated the homotropic characteristics in response to aspartate in the absence of CTP.
Corder
T S
TS
Department of Biochemistry & Biophysics, Texas A&M University System, College Station 77843.
Wild
J R
JR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D001221:Aspartate Carbamoyltransferase / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D003570:Cytidine Triphosphate / Q000378:metabolism; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D013329:Structure-Activity Relationship; D014544:Uridine Triphosphate / Q000494:pharmacology
D001224:Aspartic Acid; D003570:Cytidine Triphosphate; C013195:sparfosic acid; D000255:Adenosine Triphosphate; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid; D014544:Uridine Triphosphate