Conversion of the noncooperative Bacillus subtilis aspartate transcarbamoylase into a cooperative enzyme by a single amino acid substitution

Stebbins, Jeffrey W.; Kantrowitz, Evan R.

doi:10.1021/bi00123a017

Cited by 29 publications

(23 citation statements)

References 29 publications

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“…Therefore, the n H values could be overestimated for these two enzymes. Nevertheless, it has been observed for some enzymes that a single mutation causing decreased affinity for a ligand results in an increase in cooperativity (Stebbins and Kantrowitz, 1992;First and Fersht, 1993). The phenomenon was explained by the theory of preexisting cooperativity (First and Fersht, 1993).…”

Section: Discussionmentioning

confidence: 99%

Mutagenesis of the Glucose-1-Phosphate-Binding Site of Potato Tuber ADP-Glucose Pyrophosphorylase1

Ballícora

Preiss

1998

Plant Physiology

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Lysine (Lys)-195 in the homotetrameric ADP-glucose pyrophosphorylase (ADPGlc PPase) from Escherichia coli was shown previously to be involved in the binding of the substrate glucose-1-phosphate (Glc-1-P). This residue is highly conserved in the ADPGlc PPase family. Site-directed mutagenesis was used to investigate the function of this conserved Lys residue in the large and small subunits of the heterotetrameric potato (Solanum tuberosum) tuber enzyme. The apparent affinity for Glc-1-P of the wild-type enzyme decreased 135-to 550-fold by changing Lys-198 of the small subunit to arginine, alanine, or glutamic acid, suggesting that both the charge and the size of this residue influence Glc-1-P binding. These mutations had little effect on the kinetic constants for the other substrates (ATP and Mg 2؉ or ADP-Glc and inorganic phosphate), activator (3-phosphoglycerate), inhibitor (inorganic phosphate), or on the thermal stability. Mutagenesis of the corresponding Lys (Lys-213) in the large subunit had no effect on the apparent affinity for Glc-1-P by substitution with arginine, alanine, or glutamic acid. A double mutant, S K198R L K213R , was also obtained that had a 100-fold reduction of the apparent affinity for Glc-1-P. The data indicate that Lys-198 in the small subunit is directly involved in the binding of Glc-1-P, whereas they appear to exclude a direct role of Lys-213 in the large subunit in the interaction with this substrate.

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Section: Discussionmentioning

confidence: 99%

Mutagenesis of the Glucose-1-Phosphate-Binding Site of Potato Tuber ADP-Glucose Pyrophosphorylase1

Ballícora

Preiss

1998

Plant Physiology

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“…Cooperativity can be introduced into the E. coli ATCase catalytic trimer, B. subtilis ATCase, and E. coli OTCase by mutating Arg-105 to Ala, Arg-99 to Ala, and Arg-106 to Gly, respectively (62)(63)(64). Binding of L-ornithine to E. coli OTCase also becomes cooperative when Zn(II) binds to Cys-273 (65)(66)(67).…”

Section: Fig 3 Cartoon Drawings Of E Colimentioning

confidence: 99%

Substrate-induced conformational change in a trimeric ornithine transcarbamoylase

McCann

Tuchman

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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The crystal structure of Escherichia coli ornithine transcarbamoylase (OTCase, EC 2.1.3.3) complexed with the bisubstrate analog N-(phosphonacetyl)-L-ornithine (PALO) has been determined at 2.8-Å resolution. This research on the structure of a transcarbamoylase catalytic trimer with a substrate analog bound provides new insights into the linkages between substrate binding, protein-protein interactions, and conformational change. Anabolic ornithine transcarbamoylase (OTCase) catalyzes the first reaction in the urea cycle, in which L-ornithine is carbamoylated to form citrulline. More than 100 human mutations in OTCase that cause hyperammonemia and subsequent neurological damage or even death have been identified (1-5). Despite extensive modeling (3, 6), functional, and mutagenic studies (7,8), interpretation of the clinical results has been hampered by the unavailability of an appropriate enzyme͞ substrate analog structure.Escherichia coli anabolic OTCase (EC 2.1.3.3) shares Ϸ49% sequence similarity with its human counterpart and has a similar enzymatic mechanism (9-12). It is also homologous to the catalytic subunit of three transcarbamoylases whose crystal structures have been solved (13)(14)(15) (13), whereas E. coli ATCase has pseudo 32 symmetry with two catalytic trimers linked by three regulatory dimers (15). In contrast to B. subtilis ATCase, E. coli ATCase catalytic trimer and E. coli OTCase, which are not cooperative, the more highly aggregated transcarbamoylases are allosterically regulated (ref. 24; reviewed in refs. 25-28).The allosteric properties of E. coli ATCase have been studied most thoroughly and serve as a model for understanding other allosteric enzymes. Upon substrate binding, E. coli ATCase undergoes a large conformational change, in which the molecule expands Ϸ12 Å along its three-fold axis, each catalytic trimer and regulatory dimer rotate about the corresponding symmetry axes, the domains of the catalytic chains close about the substrate, and several interchain interfaces are restructured (20,(29)(30)(31)(32)(33). Because these changes are concerted and each chain is involved in several protein-protein interactions (33-36), short-and long-range effects are difficult to distinguish. Although the subunits have been studied extensively in solution (16,20,(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51), crystal structures of an isolated catalytic trimer of E. coli ATCase in the presence and absence of substrate analogs that would allow conformational changes restricted to the catalytic trimer to be identified have not previously been available.We report here the crystal structure of E. coli OTCase complexed with its bisubstrate analog N-(phosphonacetyl)-Lornithine (PALO) determined at 2.8-Å resolution. Active site residues that interact with the bound inhibitor are identified and the conformational change that accompanies substrate binding is described. The functional roles of specific residues are correlated with the kinetic and clinical properties of OTCase mutants and...

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“…Conceptually, it is easier to understand the creation of homotropic allosteric effects because effector and substrate are one and the same, and thus, effector affinity does not need to evolve: Only allosteric signaling needs to evolve. Accordingly, it has been shown that single point mutations can convert nonallosteric homo-oligomeric enzymes into allosteric enzymes displaying cooperativity involving their substrates (Kuo et al 1989;Scrutton et al 1992;Stebbins and Kantrowitz 1992). However, the creation of heterotropic allostery from proteins that lack effector affinity seems a more difficult task since both effector affinity and an allosteric relationship with the active site must be created.…”

mentioning

confidence: 99%

Ligand binding and allostery can emerge simultaneously

et al. 2007

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A heterotropic allosteric effect involves an effector molecule that is distinct from the substrate or ligand of the protein. How heterotropic allostery originates is an unanswered question. We have previously created several heterotropic allosteric enzymes by recombining the genes for TEM1 b-lactamase (BLA) and maltose binding protein (MBP) to create BLAs that are positively or negatively regulated by maltose. We show here that one of these engineered enzymes has ;10 6 M À1 affinity for Zn 2+, a property that neither of the parental proteins possesses. Furthermore, Zn 2+ is a negative effector that noncompetitively switches off b-lactam hydrolysis activity. Mutagenesis experiments indicate that the Zn 2+ -binding site does not involve a histidine or a cysteine, which is atypical of natural Zn 2+ -binding sites. These studies also implicate helices 1 and 12 of the BLA domain in allosteric signal propagation. These results support a model for the evolution of heterotropic allostery in which effector affinity and allosteric signaling emerge simultaneously.

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Conversion of the noncooperative Bacillus subtilis aspartate transcarbamoylase into a cooperative enzyme by a single amino acid substitution

Cited by 29 publications

References 29 publications

Mutagenesis of the Glucose-1-Phosphate-Binding Site of Potato Tuber ADP-Glucose Pyrophosphorylase1

Mutagenesis of the Glucose-1-Phosphate-Binding Site of Potato Tuber ADP-Glucose Pyrophosphorylase1

Substrate-induced conformational change in a trimeric ornithine transcarbamoylase

Ligand binding and allostery can emerge simultaneously

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