Combining multi-mutant and modular thermodynamic cycles to measure energetic coupling networks in enzyme catalysis

Carter, Charles W.; Chandrasekaran, Srinivas Niranj; Weinreb, Violetta; Li, Li; Williams, Tishan

doi:10.1063/1.4974218

Cited by 22 publications

(66 citation statements)

References 81 publications

(86 reference statements)

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“…It can be argued that the effort is fruitless as one cannot run the tape in reverse, that important and relevant witnesses are all extinct, and in particular that there is no way to test hypotheses. Our work [64,40,65,41,55,26,42,32,34,27,7,28,33,29,8,30,36,31,48], and that of others [25,66,52–54,67], tends to rebut this objection. In reality, molecular phylogenies of the Class I and II aaRS have proven to be a rich source of unexpected insights into how translation became possible and robust tools now enable us to investigate, and even recapitulate key events from the past history of life.…”

Section: Introductionmentioning

confidence: 90%

“…First, as neither intermediate construct would have sufficiently increased fitness to be selected, it suggests that the apparently separate evolutionary enhancements must have occurred coordinately, either because one of the two had already begun to function in trans , or because one or the other, or both modules could “grow” by smaller modular additions that did endow enhanced fitness [33]. Second, the modular thermodynamic cycle involving full-length TrpRS, the two distinct intermediates [33], and the Urzyme allowed measurement of a ΔΔG ‡ ~ − 5 kcal/mole coupling energy between the CP1 and anticodon-binding domains in the transition state of the amino acid activation reaction by full-length TrpRS [33], shedding new light on the general problem of intramolecular signaling or allostery [89–91]. …”

Section: Evidence For Bi-directional Coding Ancestry: Molecular Pmentioning

confidence: 99%

“…Mechanistic studies on intact TrpRS had previously identified a profound intramolecular coupling, ΔΔG ‡ ~ − 5 kcal/mole necessary for catalytic assist by the active-site Mg 2+ ion [92] and achieving full catalytic proficiency by the full-length enzyme [89,32,93,92]. A five-way coupling interaction, ΔΔG ‡ ~ − 5kcal/mole, was also measured between four residues in an allosteric switching region 20 Å from the active-site metal that mediates the shear involved in domain movement during catalysis [94].…”

Section: Evidence For Bi-directional Coding Ancestry: Molecular Pmentioning

confidence: 99%

“…A related study [32] confirmed that the same coupling energy was used in the transition state to enforce the specific selection of cognate tryptophan vs non-cognate tyrosine. Thus, the modular thermodynamic cycle provided a key link connecting the long-range coupling observed previously directly to the domain movement: the four switching side chains (I4, F26, Y33, and F37), the Mg 2+ ion, and both domains all move coordinately in the transition state [89,95]. …”

Section: Evidence For Bi-directional Coding Ancestry: Molecular Pmentioning

confidence: 99%

“…With some exceptions—TrpRS residue D132 is a Class II amino acid conserved in the amino acid substrate binding sites of several Class I aaRS—the residues that obey this particular asymmetry are located in the respective ATP binding sites, and their functional differentiation is likely to be related to functional differentiation in the mechanism for ATP activation. Class I aaRS use a dynamic Mg 2+ ion that moves with the PPi leaving group bound by the KMSKS loop during the transition state [89] and appear to stabilize additional negative charge in a dissociative transition state involving an α-metaphosphate [89]. Class II aaRS appear to stabilize a pentavalent α-phosphoryl group transition state with multiple Mg 2+ ions that are bound directly by protein residues in a manner reminiscent of the two-metal transition state stabilization of polymerases [119].…”

Section: The Basis For the Aars Class Divisionmentioning

confidence: 99%

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Coding of Class I and II Aminoacyl-tRNA Synthetases

Carter

2017

Advances in Experimental Medicine and Biology

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101

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SUMMARY The aminoacyl-tRNA synthetases and their cognate transfer RNAs translate the universal genetic code. The twenty canonical amino acids are sufficiently diverse to create a selective advantage for dividing amino acid activation between two distinct, apparently unrelated superfamilies of synthetases, Class I amino acids being generally larger and less polar, Class II amino acids smaller and more polar. Biochemical, bioinformatic, and protein engineering experiments support the hypothesis that the two Classes descended from opposite strands of the same ancestral gene. Parallel experimental deconstructions of Class I and II synthetases reveal parallel losses in catalytic proficiency at two novel modular levels—protozymes and Urzymes—associated with the evolution of catalytic activity. Bi-directional coding supports an important unification of the proteome; affords a genetic relatedness metric—middle base-pairing frequencies in sense/antisense alignments—that probes more deeply into the evolutionary history of translation than do single multiple sequence alignments; and has facilitated the analysis of hitherto unknown coding relationships in tRNA sequences. Reconstruction of native synthetases by modular thermodynamic cycles facilitated by domain engineering emphasizes the subtlety associated with achieving high specificity, shedding new light on allosteric relationships in contemporary synthetases. Synthetase Urzyme structural biology suggests that they are catalytically active molten globules, broadening the potential manifold of polypeptide catalysts accessible to primitive genetic coding and motivating revisions of the origins of catalysis. Finally, bi-directional genetic coding of some of the oldest genes in the proteome places major limitations on the likelihood that any RNA World preceded the origins of coded proteins.

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Section: Introductionmentioning

confidence: 90%

Section: Evidence For Bi-directional Coding Ancestry: Molecular Pmentioning

confidence: 99%

Section: Evidence For Bi-directional Coding Ancestry: Molecular Pmentioning

confidence: 99%

Section: Evidence For Bi-directional Coding Ancestry: Molecular Pmentioning

confidence: 99%

Section: The Basis For the Aars Class Divisionmentioning

confidence: 99%

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Coding of Class I and II Aminoacyl-tRNA Synthetases

Carter

2017

Advances in Experimental Medicine and Biology

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101

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Escapement mechanisms: Efficient free energy transduction by reciprocally‐coupled gating

Carter

2019

Proteins

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Conversion of the free energy of NTP hydrolysis efficiently into mechanical work and/or information by transducing enzymes sustains living systems far from equilibrium, and so has been of interest for many decades. Detailed molecular mechanisms, however, remain puzzling and incomplete. We previously reported that catalysis of tryptophan activation by tryptophanyl‐tRNA synthetase, TrpRS, requires relative domain motion to re‐position the catalytic Mg2+ ion, noting the analogy between that conditional hydrolysis of ATP and the escapement mechanism of a mechanical clock. The escapement allows the time‐keeping mechanism to advance discretely, one gear at a time, if and only if the pendulum swings, thereby converting energy from the weight driving the pendulum into rotation of the hands. Coupling of catalysis to domain motion, however, mimics only half of the escapement mechanism, suggesting that domain motion may also be reciprocally coupled to catalysis, completing the escapement metaphor. Computational studies of the free energy surface restraining the domain motion later confirmed that reciprocal coupling: the catalytic domain motion is thermodynamically unfavorable unless the PPi product is released from the active site. These two conditional phenomena—demonstrated together only for the TrpRS mechanism—function as reciprocally‐coupled gates. As we and others have noted, such an escapement mechanism is essential to the efficient transduction of NTP hydrolysis free energy into other useful forms of mechanical or chemical work and/or information. Some implementation of both gating mechanisms—catalysis by domain motion and domain motion by catalysis—will thus likely be found in many other systems.

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Structural insights into the specific interaction between Geobacillus stearothermophilus tryptophanyl-tRNA synthetase and antimicrobial Chuangxinmycin

Fan

Feng

et al. 2022

Journal of Biological Chemistry

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show abstract

Combining multi-mutant and modular thermodynamic cycles to measure energetic coupling networks in enzyme catalysis

Cited by 22 publications

References 81 publications

Coding of Class I and II Aminoacyl-tRNA Synthetases

Coding of Class I and II Aminoacyl-tRNA Synthetases

Escapement mechanisms: Efficient free energy transduction by reciprocally‐coupled gating

Structural insights into the specific interaction between Geobacillus stearothermophilus tryptophanyl-tRNA synthetase and antimicrobial Chuangxinmycin

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