scholarly journals Assessment of the allosteric mechanism of aspartate transcarbamoylase based on the crystalline structure of the unregulated catalytic subunit

1999 ◽  
Vol 96 (10) ◽  
pp. 5388-5393 ◽  
Author(s):  
P. T. Beernink ◽  
J. A. Endrizzi ◽  
T. Alber ◽  
H. K. Schachman
Keyword(s):  
Crystalline Structure ◽  
Catalytic Subunit ◽  
Aspartate Transcarbamoylase ◽  
Allosteric Mechanism

Hybrid aspartate transcarbamoylase containing cross-linked subunits

1981 ◽  
Vol 59 (6) ◽  
pp. 461-468 ◽  
Author(s):  
William W.-C. Chan ◽  
Caroline A. Enns
Keyword(s):  
Conformational Changes ◽  
Catalytic Subunit ◽  
Aspartate Transcarbamoylase ◽  
Regulatory Subunit ◽  
Cross Linking ◽  
Substrate Affinity ◽  
Catalytic Subunits ◽  
Regulatory Subunits ◽  
Allosteric Mechanism ◽  
Enzyme Molecules

The role of conformational changes and subunit interactions in the allosteric mechanism of aspartate transcarbamoylase was evaluated by studying hybrid enzyme molecules containing cross-linked subunits. Native enzyme was cross-linked with tartryl diazide in the presence and absence of substrate analogues. The two types of modified enzyme derivatives were each dissociated into catalytic (c3) and regulatory (r2) subunits. Hybrids were constructed with modified catalytic subunits and unmodified regulatory subunits or vice versa. Subunits from different derivatives also formed hybrids.All hybrids containing cross-linked catalytic subunits showed hyperbolic substrate saturation curves while cross-linking in the regulatory subunit alone did not abolish cooperativity. The type of cross-linking in the catalytic subunit had a decisive influence on the substrate affinity of the hybrid as well as its response to the allosteric effectors ATP and CTP. However many effects were also dependent on the presence of regulatory subunits. The results implicate a substantial conformational change in the catalytic subunit upon substrate binding and suggest an important role for the c–r interaction in the allosteric mechanism.

scholarly journals Effects of lipids on nucleotide inhibition of wheat-germ aspartate transcarbamoylase: evidence of an additional level of control?

1996 ◽  
Vol 313 (2) ◽  
pp. 669-673 ◽  
Author(s):  
Ashan KHAN ◽  
Babur Z. CHOWDHRY ◽  
Robert J. YON
Keyword(s):  
Fatty Acids ◽  
Wheat Germ ◽  
Hill Coefficient ◽  
Aspartate Transcarbamoylase ◽  
Kinetic Effect ◽  
Carbamoyl Phosphate ◽  
Allosteric Mechanism ◽  
Additional Level ◽  
Nucleotide Inhibition ◽  
Metabolic Significance

Wheat-germ aspartate transcarbamoylase, a monofunctional trimer, is strongly inhibited by uridine 5ʹ-monophosphate (UMP), which shows kinetic interactions with the substrate, carbamoyl phosphate, suggesting a classical allosteric mechanism of regulation. Inhibition of the purified enzyme by UMP was amplified in the presence of a variety of ionic lipids at concentrations low enough to preclude denaturation. In the absence of UMP, most of these compounds had no kinetic effect or were slightly activating. Two phospholipids did not show the effect. In a homologous series of fatty acids (C6-C16), the potentiating effect was only seen with homologues greater than C8, reaching a maximum at C12. The effect of dodecanoate (C12) on kinetic cooperativity (UMP as variable ligand) was studied. At each of several fixed concentrations of carbamoyl phosphate, dodecanoate had a pronounced effect on the half-saturating concentration of UMP, which was reduced by about half in every case, indicating substantially tighter binding of UMP. However, dodecanoate had relatively little effect on the kinetic Hill coefficient for the cooperativity of UMP. The possible metabolic significance of these effects is discussed.

Structure and function of aspartate transcarbamoylase studied using chymotrypsin as a probe

1978 ◽  
Vol 56 (6) ◽  
pp. 654-658 ◽  
Author(s):  
William W.-C. Chan ◽  
Caroline A. Enns
Keyword(s):  
Active Sites ◽  
Sodium Dodecyl ◽  
Peptide Bond ◽  
Structure And Function ◽  
Catalytic Subunit ◽  
Native Enzyme ◽  
Aspartate Transcarbamoylase ◽  
Carbamoyl Phosphate ◽  
And Function ◽  
Extended Exposure

Aspartate transcarbamoylase from Escherichia coli is composed of six catalytic (c) and six regulatory (r) polypeptides. We have studied the structure and function of this enzyme using chymotrypsin as a probe. The protease inactivates the isolated catalytic subunit (c3) but has no effects on the native enzyme (c6r6). Under identical conditions, the c3r6 complex is inactivated at a much slower rate than c3. The presence of the substrate analogue succinate together with carbamoyl phosphate reduces substantially the rate of inactivation. Extended exposure to chymotrypsin converts the catalytic subunit into a partially active derivative with a fourfold higher Michaelis constant. This derivative is indistinguishable from the unmodified catalytic subunit in gel electrophoresis under nondenaturing conditions. However, in the presence of sodium dodecyl sulfate, the major fragment in the electropherogram is smaller than that of the intact catalytic polypeptide. The results could be explained by postulating the presence of a chymotrypsin-sensitive peptide bond at or near the active site. Since X-ray crystallographic studies have indicated that the active sites are located in a central cavity, the resistance of the native enzyme towards inactivation may be due to the inability of chymotrypsin to enter this cavity.

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