Arginine 54 in the active site of escherichia coli aspartate transcarbamoylase is critical for catalysis: A site‐specific mutagenesis, NMR, and X‐ray crystallographic study

Stebbins, Jeffrey W.; Robertson, Diane E.; Roberts, Mary F.; Stevens, Raymond C.; Lipscomb, William N.; Kantrowitz, Evan R.

doi:10.1002/pro.5560011105

Cited by 23 publications

(26 citation statements)

References 43 publications

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“…The localization of the N-terminal region of the regulatory chain (residues 1–7) was first modeled in the CTP-bound complex 40 near the allosteric site. 38 Mutagenesis studies 41 support the suggestion that this is the regulatory region of the enzyme. Increased resolution of the X-ray data, 2.1Å 23 versus 2.5Å, 40 yielded electron density for the independent r1 and r6 chains and allowed tracing of the first seven residues, and remodeling residues 8r–10r.…”

Section: Heterotropic Effects and Structural Aspectsmentioning

confidence: 88%

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Structure and Mechanisms of Escherichia coli Aspartate Transcarbamoylase

Lipscomb

Kantrowitz

2011

Acc. Chem. Res.

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Enzymes catalyze a particular reaction in cells, but only a few control the rate of this reaction and the metabolic pathway that follows. One specific mechanism for such enzymatic control of a metabolic pathway involves molecular feedback, whereby a metabolite further down the pathway acts at a unique site on the control enzyme to alter its activity allosterically. This regulation may be positive or negative (or both), depending upon the particular system. Another method of enzymatic control involves the cooperative binding of the substrate, which allows a large change in enzyme activity to emanate from only a small change in substrate concentration. Allosteric regulation and homotropic cooperativity are often known to involve significant conformational changes in the structure of the protein. Escherichia coli aspartate transcarbamoylase (ATCase) is the textbook example of an enzyme that regulates a metabolic pathway, namely, pyrimidine nucleotide biosynthesis, by feedback control and by the cooperative binding of the substrate, l-aspartate. The catalytic and regulatory mechanisms of this enzyme have been extensively studied. A series of X-ray crystal structures of the enzyme in the presence and absence of substrates, products, and analogues have provided details, at the molecular level, of the conformational changes that the enzyme undergoes as it shifts between its low-activity, low-affinity form (T state) to its high-activity, high-affinity form (R state). These structural data provide insights into not only how this enzyme catalyzes the reaction between l-aspartate and carbamoyl phosphate to form N-carbamoyl-l-aspartate and inorganic phosphate, but also how the allosteric effectors modulate this activity. In this Account, we summarize studies on the structure of the enzyme and describe how these structural data provide insights into the catalytic and regulatory mechanisms of the enzyme. The ATCase-catalyzed reaction is regulated by nucleotide binding some 60 Å from the active site, inducing structural alterations that modulate catalytic activity. The delineation of the structure and function in this particular model system will help in understanding the molecular basis of cooperativity and allosteric regulation in other systems as well.

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Section: Heterotropic Effects and Structural Aspectsmentioning

confidence: 88%

“…In support of the important role of Arg54, when Arg54 is replaced by Ala enzymatic activity is reduced by a factor of about 17,000 fold. 37,38 …”

Section: The Active Site and Mechanistic Aspectsmentioning

confidence: 99%

Structure and Mechanisms of Escherichia coli Aspartate Transcarbamoylase

Lipscomb

Kantrowitz

2011

Acc. Chem. Res.

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“…In both cases x-ray structures of the mutant holoenzymes have been determined in the presence of PALA. The structure of the R54A holoenzyme in the presence of PALA is virtually identical to that of the wild-type R state except at the site of the amino acid substitution (6). The results for the R105A holoenzyme in the presence of PALA are much different.…”

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confidence: 83%

Importance of Domain Closure for the Catalysis and Regulation ofEscherichia coli Aspartate Transcarbamoylase

Macol¹,

Tsuruta²,

Kantrowitz³

2002

Journal of Biological Chemistry

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Two hybrid versions of Escherichia coli aspartate transcarbamoylase were studied to determine the influence of domain closure on the homotropic and heterotropic properties of the enzyme. Each hybrid holoenzyme had one wild-type and one inactive catalytic subunit. In the first case the inactive catalytic subunit had Arg-54 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 17,000-fold reduction in activity, no loss in substrate affinity, and an R state structurally identical to that of the wild-type enzyme. In the second case, the inactive catalytic subunit had Arg-105 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 1,100-fold reduction in activity, substantial loss in substrate affinity, and loss of the ability to be converted to the R state. Thus, the R54A substitution results in a holoenzyme that can undergo closure of the catalytic chain domains to form the high activity, high affinity active site and to undergo the allosteric transition, whereas the R105A substitution results in a holoenzyme that can neither undergo domain closure nor the allosteric transition. The hybrid holoenzyme with one wild-type and one R54A catalytic subunit exhibited the same maximal velocity per active site as the wild-type holoenzyme, reduced cooperativity, and normal heterotropic interactions. The hybrid with one wild-type and one R105A catalytic subunit exhibited significantly reduced maximal velocity per active site as compared with the wild-type holoenzyme, reduced cooperativity, and substantially reduced heterotropic interactions. Small angle x-ray scattered was used to verify that the R105A-containing hybrid could attain an R state structure. These results indicate the global nature of the conformational changes associated with the allosteric transition in the enzyme. If one catalytic subunit cannot undergo domain closure to create the active sites, then the entire molecule cannot attain the high activity, high activity R state.The homotropic cooperativity in Escherichia coli aspartate transcarbamoylase (EC 2.1.3.2) is directly related to the ability of the substrates to induce a structural and functional transition of the enzyme from a low activity low affinity T state to a high activity, high affinity R state (1). Structurally the conversion of the enzyme from the T to the R state involves an elongation of the enzyme by 11 Å along the molecular 3-fold axis along with a twist of the two catalytic subunits of about 5°in opposite directions (2). This structural transition also results in a simultaneous rotation of each regulatory dimer about its own 2-fold axis. In addition to these quaternary structural changes, the T to R transition involves structural alterations on the tertiary level. For example, during the T to R transition each catalytic chain undergoes a domain closure in which the aspartate domain moves 3 Å toward the carbamoyl phosphate domain, resulting in a major reorganization of the interdomain interface. In addition to this ...

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“…It has been shown that a mutation of this residue to Ala in E. coli ATC caused a 17,000-fold decrease in the enzyme activity. 25,26 This arginine is Fig. 3.…”

Section: Active Sitementioning

confidence: 98%