scholarly journals N 6-Methyladenosines in mRNAs reduce the accuracy of codon reading by transfer RNAs and peptide release factors

2021 ◽  
Vol 49 (5) ◽  
pp. 2684-2699
Author(s):  
Ka-Weng Ieong ◽  
Gabriele Indrisiunaite ◽  
Arjun Prabhakar ◽  
Joseph D Puglisi ◽  
Måns Ehrenberg
Keyword(s):  
Single Molecule ◽  
Stop Codon ◽  
Product Formation ◽  
Release Factors ◽  
Substrate Complex ◽  
Peptidyl Transfer ◽  
Enzyme Substrate ◽  
Peptide Release ◽  
Accuracy Ratio ◽  
Codon Reading

Abstract We used quench flow to study how N6-methylated adenosines (m6A) affect the accuracy ratio between kcat/Km (i.e. association rate constant (ka) times probability (Pp) of product formation after enzyme-substrate complex formation) for cognate and near-cognate substrate for mRNA reading by tRNAs and peptide release factors 1 and 2 (RFs) during translation with purified Escherichia coli components. We estimated kcat/Km for Glu-tRNAGlu, EF-Tu and GTP forming ternary complex (T3) reading cognate (GAA and Gm6AA) or near-cognate (GAU and Gm6AU) codons. ka decreased 10-fold by m6A introduction in cognate and near-cognate cases alike, while Pp for peptidyl transfer remained unaltered in cognate but increased 10-fold in near-cognate case leading to 10-fold amino acid substitution error increase. We estimated kcat/Km for ester bond hydrolysis of P-site bound peptidyl-tRNA by RF2 reading cognate (UAA and Um6AA) and near-cognate (UAG and Um6AG) stop codons to decrease 6-fold or 3-fold by m6A introduction, respectively. This 6-fold effect on UAA reading was also observed in a single-molecule termination assay. Thus, m6A reduces both sense and stop codon reading accuracy by decreasing cognate significantly more than near-cognate kcat/Km, in contrast to most error inducing agents and mutations, which increase near-cognate at unaltered cognate kcat/Km.

scholarly journals Proofreading through spatial gradients

2020 ◽  
Author(s):  
Vahe Galstyan ◽  
Kabir Husain ◽  
Fangzhou Xiao ◽  
Arvind Murugan ◽  
Rob Phillips
Keyword(s):  
Error Correction ◽  
Reaction Rates ◽  
Structural Features ◽  
Product Formation ◽  
Concentration Gradients ◽  
Nucleotide Hydrolysis ◽  
Substrate Complex ◽  
Spatial Gradients ◽  
Enzyme Substrate ◽  
Kinetic Proofreading

Key enzymatic processes in biology use the nonequilibrium error correction mechanism called kinetic proofreading to enhance their specificity. Kinetic proofreading typically requires several dedicated structural features in the enzyme, such as a nucleotide hydrolysis site and multiple enzyme–substrate conformations that delay product formation. Such requirements limit the applicability and the adaptability of traditional proofreading schemes. Here, we explore an alternative conceptual mechanism of error correction that achieves delays between substrate binding and subsequent product formation by having these events occur at distinct physical locations. The time taken by the enzyme–substrate complex to diffuse from one location to another is leveraged to discard wrong substrates. This mechanism does not require dedicated structural elements on the enzyme, making it easier to overlook in experiments but also making proofreading tunable on the fly. We discuss how tuning the length scales of enzyme or substrate concentration gradients changes the fidelity, speed and energy dissipation, and quantify the performance limitations imposed by realistic diffusion and reaction rates in the cell. Our work broadens the applicability of kinetic proofreading and sets the stage for the study of spatial gradients as a possible route to specificity.

scholarly journals Probing conformational dynamics of an enzymatic active site by an in situ single fluorogenic probe under piconewton force manipulation

2016 ◽  
Vol 113 (52) ◽  
pp. 15006-15011 ◽  
Author(s):  
Nibedita Pal ◽  
Meiling Wu ◽  
H. Peter Lu
Keyword(s):  
Single Molecule ◽  
Active Site ◽  
Complex Dynamics ◽  
Conformational Dynamics ◽  
Enzymatic Reaction ◽  
Product Formation ◽  
Magnetic Tweezers ◽  
Complex Nature ◽  
Enzyme Substrate ◽  
Multiple Product

Unraveling the conformational details of an enzyme during the essential steps of a catalytic reaction (i.e., enzyme–substrate interaction, enzyme–substrate active complex formation, nascent product formation, and product release) is challenging due to the transient nature of intermediate conformational states, conformational fluctuations, and the associated complex dynamics. Here we report our study on the conformational dynamics of horseradish peroxidase using single-molecule multiparameter photon time-stamping spectroscopy with mechanical force manipulation, a newly developed single-molecule fluorescence imaging magnetic tweezers nanoscopic approach. A nascent-formed fluorogenic product molecule serves as a probe, perfectly fitting in the enzymatic reaction active site for probing the enzymatic conformational dynamics. Interestingly, the product releasing dynamics shows the complex conformational behavior with multiple product releasing pathways. However, under magnetic force manipulation, the complex nature of the multiple product releasing pathways disappears and more simplistic conformations of the active site are populated.

scholarly journals The pre-eminence of kcat. in the manifestation of optimal enzymic activity delineated by using the Briggs-Haldane two-step irreversible kinetic model

1976 ◽  
Vol 159 (1) ◽  
pp. 165-166 ◽  
Author(s):  
K Brocklehurst ◽  
A Cornish-Bowden
Keyword(s):  
Kinetic Model ◽  
Dissociation Constant ◽  
Substrate Concentration ◽  
Product Formation ◽  
Enzymic Activity ◽  
Substrate Complex ◽  
Diffusion Limited ◽  
Enzyme Substrate

The suggestion by Fersht [(1974) Proc. R. Soc. London Ser. B 187, 397-407] that enzymes that provide maximal rates of catalysis should be characterized by values of Ks, the dissociation constant of the enzyme-substrate complex, greater than 10 times the value of the ambient substrate concentration has been examined. 2. For such enzymes, Ks is not relevant, and attention is best focused on the relative numerical values of k(cat). (in units of s(-1) and the substrate molarity. It is necessary only that the former be about 10(10)-10(11) times the latter to ensure that the rate of product formation be diffusion-limited and thus maximal.

scholarly journals Dynamics of ribosomes and release factors during translation termination in E. coli

2018 ◽  
Author(s):  
Sarah Adio ◽  
Heena Sharma ◽  
Tamara Senyushkina ◽  
Prajwal Karki ◽  
Cristina Maracci ◽  
...  
Keyword(s):  
Single Molecule ◽  
Translation Termination ◽  
Bound State ◽  
Gtp Hydrolysis ◽  
E Coli ◽  
Release Factors ◽  
Single Molecule Fret ◽  
Peptide Release ◽  
Two Factors ◽  
Stochastic Assembly

AbstractRelease factors RF1 and RF2 promote hydrolysis of peptidyl-tRNA during translation termination. The GTPase RF3 promotes recycling of RF1 and RF2. Using single molecule FRET together with ensemble kinetics, we show that ribosome termination complexes that carry two factors, RF1–RF3 or RF2–RF3, are dynamic and fluctuate between non-rotated and rotated states, while each factor alone has its distinct signature on the ribosome dynamics and conformation. Dissociation of RF1 depends on peptide release and the presence of RF3, whereas RF2 can dissociate spontaneously. RF3 binds in the GTP-bound state and can rapidly dissociate without GTP hydrolysis from termination complex carrying RF1. GTP cleavage helps RF3 release from ribosomes stalled in the rotated state in the absence of RF1. Our data suggest how the stochastic assembly of the ribosome–RF1–RF3–GTP complex, peptide release, and ribosome fluctuations promote termination of protein synthesis and recycling of the release factors.

scholarly journals Dynamics of ribosomes and release factors during translation termination in E. coli

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Sarah Adio ◽  
Heena Sharma ◽  
Tamara Senyushkina ◽  
Prajwal Karki ◽  
Cristina Maracci ◽  
...  
Keyword(s):  
Single Molecule ◽  
Translation Termination ◽  
Bound State ◽  
Gtp Hydrolysis ◽  
E Coli ◽  
Release Factors ◽  
Single Molecule Fret ◽  
Biochemical Assays ◽  
Peptide Release ◽  
Two Factors

Release factors RF1 and RF2 promote hydrolysis of peptidyl-tRNA during translation termination. The GTPase RF3 promotes recycling of RF1 and RF2. Using single molecule FRET and biochemical assays, we show that ribosome termination complexes that carry two factors, RF1–RF3 or RF2–RF3, are dynamic and fluctuate between non-rotated and rotated states, whereas each factor alone has its distinct signature on ribosome dynamics and conformation. Dissociation of RF1 depends on peptide release and the presence of RF3, whereas RF2 can dissociate spontaneously. RF3 binds in the GTP-bound state and can rapidly dissociate without GTP hydrolysis from termination complex carrying RF1. In the absence of RF1, RF3 is stalled on ribosomes if GTP hydrolysis is blocked. Our data suggest how the assembly of the ribosome–RF1–RF3–GTP complex, peptide release, and ribosome fluctuations promote termination of protein synthesis and recycling of the release factors.

scholarly journals Derivable Equations and Issues Often Ignored in the Original Michaelis-Menten Mathematical Formalism

2019 ◽  
pp. 1-13
Author(s):  
Ikechukwu I. Udema
Keyword(s):  
Rate Constant ◽  
Product Formation ◽  
Theoretical Research ◽  
Quasi Steady State ◽  
Substrate Complex ◽  
The Core ◽  
Enzyme Substrate ◽  
Reverse Rate Constant ◽  
Core Issue

Background: There has been recent shift from the core issue of Michaelian kinetics to issues regarding various kinds of quasi-steady-state assumptions. Derivable equations with which to determine reverse rate constant for the dissociation of enzyme-substrate complex (ES) is given less attention. Objectives: The objectives of this research are: 1) to derive other equations from differential equations whose evaluation leads to MM equation and 2) quantify based on derived equations the kinetic parameters given less attention and duration of catalytic events. Methods: A major theoretical research and experimentation using Bernfeld method. Results and Discussion: The durations for ES dissociation (ESD) into free substrate, S and enzyme, E were much shorter than the duration of ESD into E and product, P in 3 minutes duration of assay with low [S]; it was the shortest and longest in 3 and 5 minutes durations respectively with high [S]. The durations of ESD into E and P was shortest in 3 minutes duration of assay with high [S]. The values of reverse rate constant, k-1 for ESD into S and E in 3 minutes duration of assay with high [S] was » the rate constant, k2 for product formation and they are much higher than in other duration of assay. Conclusion: The equations for the determination of the durations of various events, in a given catalytic cycle were derived. The various time regimes for each event and the rate constant for the dissociation of the ES can be graphically and calculationally determined as the case may be. Substrate concentration regime and duration of assay affects rate constants.

scholarly journals Proofreading through spatial gradients

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Vahe Galstyan ◽  
Kabir Husain ◽  
Fangzhou Xiao ◽  
Arvind Murugan ◽  
Rob Phillips
Keyword(s):  
Error Correction ◽  
Reaction Rates ◽  
Structural Features ◽  
Product Formation ◽  
Nucleotide Hydrolysis ◽  
Structural Requirements ◽  
Substrate Complex ◽  
Spatial Gradients ◽  
Enzyme Substrate ◽  
Kinetic Proofreading

Key enzymatic processes use the nonequilibrium error correction mechanism called kinetic proofreading to enhance their specificity. The applicability of traditional proofreading schemes, however, is limited since they typically require dedicated structural features in the enzyme, such as a nucleotide hydrolysis site or multiple intermediate conformations. Here, we explore an alternative conceptual mechanism that achieves error correction by having substrate binding and subsequent product formation occur at distinct physical locations. The time taken by the enzyme-substrate complex to diffuse from one location to another is leveraged to discard wrong substrates. This mechanism does not have the typical structural requirements, making it easier to overlook in experiments. We discuss how the length scales of molecular gradients dictate proofreading performance, and quantify the limitations imposed by realistic diffusion and reaction rates. Our work broadens the applicability of kinetic proofreading and sets the stage for studying spatial gradients as a possible route to specificity.

The role of secondary alcoholic groups of D-galacturonan in its degradation with exo-D-galacturonanase

1980 ◽  
Vol 45 (2) ◽  
pp. 427-434 ◽  
Author(s):  
Kveta Heinrichová ◽  
Rudolf Kohn
Keyword(s):  
Acetyl Derivative ◽  
Competitive Inhibitor ◽  
Hydroxyl Groups ◽  
Pectic Acid ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Individual ◽  
Degradation Degree ◽  
Derivatives Of

The effect of exo-D-galacturonanase from carrot on O-acetyl derivatives of pectic acid of variousacetylation degree was studied. Substitution of hydroxyl groups at C(2) and C(3) of D-galactopyranuronic acid units influences the initial rate of degradation, degree of degradation and its maximum rate, the differences being found also in the time of limit degradations of the individual O-acetyl derivatives. Value of the apparent Michaelis constant increases with increase of substitution and value of Vmax changes. O-Acetyl derivatives act as a competitive inhibitor of degradation of D-galacturonan. The extent of the inhibition effect depends on the degree of substitution. The only product of enzymic reaction is D-galactopyranuronic acid, what indicates that no degradation of the terminal substituted unit of O-acetyl derivative of pectic acid takes place. Substitution of hydroxyl groups influences the affinity of the enzyme towards the modified substrate. The results let us presume that hydroxyl groups at C(2) and C(3) of galacturonic unit of pectic acid are essential for formation of the enzyme-substrate complex.

scholarly journals Spontaneous Cleavages of a Heterologous Protein, the CenA Endoglucanase of Cellulomonas fimi, in Escherichia coli

2021 ◽  
Vol 14 ◽  
pp. 117863612110246
Author(s):  
Cheuk Yin Lai ◽  
Ka Lun Ng ◽  
Hao Wang ◽  
Chui Chi Lam ◽  
Wan Keung Raymond Wong
Keyword(s):  
Escherichia Coli ◽  
Cellulolytic Bacterium ◽  
Systematic Screening ◽  
Cellulomonas Fimi ◽  
E Coli ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Glycosylation Sites ◽  
Endogenous Proteins ◽  
Cellulose Binding

CenA is an endoglucanase secreted by the Gram-positive cellulolytic bacterium, Cellulomonas fimi, to the environment as a glycosylated protein. The role of glycosylation in CenA is unclear. However, it seems not crucial for functional activity and secretion since the unglycosylated counterpart, recombinant CenA (rCenA), is both bioactive and secretable in Escherichia coli. Using a systematic screening approach, we have demonstrated that rCenA is subjected to spontaneous cleavages (SC) in both the cytoplasm and culture medium of E. coli, under the influence of different environmental factors. The cleavages were found to occur in both the cellulose-binding (CellBD) and catalytic domains, with a notably higher occurring rate detected in the former than the latter. In CellBD, the cleavages were shown to occur close to potential N-linked glycosylation sites, suggesting that these sites might serve as ‘attributive tags’ for differentiating rCenA from endogenous proteins and the points of initiation of SC. It is hypothesized that glycosylation plays a crucial role in protecting CenA from SC when interacting with cellulose in the environment. Subsequent to hydrolysis, SC would ensure the dissociation of CenA from the enzyme-substrate complex. Thus, our findings may help elucidate the mechanisms of protein turnover and enzymatic cellulolysis.

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