Immunological studies of aspartate transcarbamoylase. I. Characterizat ion of the native enzyme, catalytic, and regulatory subunit immune systems

Biochemistry ◽  
1968 ◽  
Vol 7 (12) ◽  
pp. 4315-4322 ◽  
Author(s):  
Martha R. Bethell ◽  
Roland. Von Fellenberg ◽  
Mary Ellen. Jones ◽  
Lawrence. Levine
Keyword(s):  
Native Enzyme ◽  
Aspartate Transcarbamoylase ◽  
Regulatory Subunit ◽  
Immune Systems

scholarly journals Conformational Plasticity of the Active Site Entrance in E. coli Aspartate Transcarbamoylase and Its Implication in Feedback Regulation

2020 ◽  
Vol 21 (1) ◽  
pp. 320
Author(s):  
Zhen Lei ◽  
Nan Wang ◽  
Hongwei Tan ◽  
Jimin Zheng ◽  
Zongchao Jia
Keyword(s):  
Active Site ◽  
Feedback Regulation ◽  
Aspartate Transcarbamoylase ◽  
Regulatory Subunit ◽  
Dynamic Simulations ◽  
E Coli ◽  
Open Conformation ◽  
Conformational Plasticity ◽  
The Stability ◽  
Remote Feedback

Aspartate transcarbamoylase (ATCase) has been studied for decades and Escherichia coli ATCase is referred as a “textbook example” for both feedback regulation and cooperativity. However, several critical questions about the catalytic and regulatory mechanisms of E. coli ATCase remain unanswered, especially about its remote feedback regulation. Herein, we determined a structure of E. coli ATCase in which a key residue located (Arg167) at the entrance of the active site adopted an uncommon open conformation, representing the first wild-type apo-form E. coli ATCase holoenzyme that features this state. Based on the structure and our results of enzymatic characterization, as well as molecular dynamic simulations, we provide new insights into the feedback regulation of E. coli ATCase. We speculate that the binding of pyrimidines or purines would affect the hydrogen bond network at the interface of the catalytic and regulatory subunit, which would further influence the stability of the open conformation of Arg167 and the enzymatic activity of ATCase. Our results not only revealed the importance of the previously unappreciated open conformation of Arg167 in the active site, but also helped to provide rationalization for the mechanism of the remote feedback regulation of ATCase.

Further studies on aspartate transcarbamoylase: molecular weight of the c3r6 complex and analysis of succinate inhibition in the native enzyme

1976 ◽  
Vol 54 (12) ◽  
pp. 1061-1068
Author(s):  
William W.-C. Chan
Keyword(s):  
Molecular Weight ◽  
Competitive Inhibition ◽  
Gel Filtration ◽  
Frictional Coefficient ◽  
Sedimentation Coefficient ◽  
Native Enzyme ◽  
Aspartate Transcarbamoylase ◽  
Unusual Structure ◽  
Regulatory Subunits ◽  
Method Of Calculation

The complex which is formed when excess regulatory subunits (r2) of aspartate transcarbamoylase (EC 2.1.3.2) are added to a dilute solution of the catalytic subunit (c3) has been studied by gel-filtration on Sephadex G-200. The elution volume indicates a Stokes' radius of between 5.42 and 5.92 nm, depending on the method of calculation. Using the sedimentation coefficient of 7.7 S previously determined, the molecular weight is estimated to be close to 200 000, in support of the c3r6 structure proposed earlier for the complex. The calculated frictional coefficient indicates abnormal hydrodynamic properties which are probably due to unusual structure characteristics.The pattern of succinate inhibition of native aspartate transcarbamoylase has also been analyzed. At low concentrations, succinate activates the enzyme, presumably by converting it from the taut state to the relaxed (R) state. Further increase in the succinate concentration leads to competitive inhibition of the R state. Using a novel procedure for analysis of the data, the Michaelis constant for aspartate of the R state has been estimated to be about 7 mM. This value is close to the Km of c3r6 for aspartate, measured under identical conditions. The result therefore provides further evidence suggesting that the c3r6 complex resembles the R state of the native enzyme.

scholarly journals Aspartate Transcarbamoylase Molecules Lacking One Regulatory Subunit

1974 ◽  
Vol 71 (3) ◽  
pp. 918-922 ◽  
Author(s):  
Y. R. Yang ◽  
J. M. Syvanen ◽  
G. M. Nagel ◽  
H. K. Schachman
Keyword(s):  
Aspartate Transcarbamoylase ◽  
Regulatory Subunit

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.

Conformation of cross-linked aspartate transcarbamoylase

1981 ◽  
Vol 59 (5) ◽  
pp. 371-378 ◽  
Author(s):  
William W.-C. Chan
Keyword(s):  
Conformational Change ◽  
Conformational Flexibility ◽  
The Other ◽  
Native Enzyme ◽  
Aspartate Transcarbamoylase ◽  
Cross Linking ◽  
Aspartate Transcarbamylase ◽  
Substrate Analogues ◽  
T State ◽  
Substrate Concentrations

We have previously shown that aspartate transcarbamylase loses its substrate cooperativity after modification with a cross-linking reagent. Depending on the presence or absence of substrate analogues during cross-linking, the derivatives resemble the relaxed (R) or taut (T) state, respectively. In the present study, we attempt to characterize the conformation of these derivatives and the effects of ligands.The putative T-state derivative was similar to the native enzyme in its reactivity towards p-hydroxymercuribenzoate and in the increase of reactivity upon addition of succinate. However, unlike the native enzyme it was not activated by succinate at low substrate concentrations. On the other hand, the putative R-state derivative showed greatly enhanced reactivity which was not substantially increased by succinate. In the presence of urea, the native enzyme and the two cross-linked derivatives all resembled the R state. Thus at low substrate concentrations urea activated both the native enzyme and the T-state derivative. In contrast, the effect of urea on the R state derivative is mainly inhibitory.The above results show that the R state has been definitely stabilized whereas the T-state derivative retains some conformational flexibility. Our observations also indicate that the conformational change induced by succinate has two distinct components of which only one is allowed in the T-state derivative.

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.

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