Pre-steady State Research Articles

Pre-steady-state Kinetic Analysis of Amino Acid Transporter SLC6A14 Reveals Rapid Turnover Rate and Substrate Translocation.

SLC6A14 (solute carrier family 6 member 14) is an amino acid transporter, driven by Na+ and Cl− co-transport, whose structure, function, and molecular and kinetic mechanism have not been well characterized. Its broad substrate selectivity, including neutral and cationic amino acids, differentiates it from other SLC6 family members, and its proposed involvement in nutrient transport in several cancers suggest that it could become an important drug target. In the present study, we investigated SLC6A14 function and its kinetic mechanism after expression in human embryonic kidney (HEK293) cells, including substrate specificity and voltage dependence under various ionic conditions. We applied rapid solution exchange, voltage jumps, and laser photolysis of caged alanine, allowing sub-millisecond temporal resolution, to study SLC6A14 steady state and pre-steady state kinetics. The results highlight the broad substrate specificity and suggest that extracellular chloride enhances substrate transport but is not required for transport. As in other SLC6 family members, Na+ binding to the substrate-free transporter (or conformational changes associated with it) is electrogenic and is likely rate limiting for transporter turnover. Transient current decaying with a time constant of <1ms is also observed after rapid amino acid application, both in forward transport and homoexchange modes, indicating a slightly electrogenic, but fast and not rate-limiting substrate translocation step. Our results, which are consistent with kinetic modeling, suggest rapid transporter turnover rate and substrate translocation with faster kinetics compared with other SLC6 family members. Together, these results provided novel information on the SLC6A14 transport cycle and mechanism, expanding our understanding of SLC6A14 function.

SSM-based electrophysiology, a label-free real-time method reveals sugar binding & transport events in SGLT1

Here, we present a solid-supported membrane (SSM)-based electrophysiological approach to study sugar binding and Na+/glucose cotransport by SGLT1 in membrane vesicles. SSM-based electrophysiology delivers a cumulative real-time current readout from numerous SGLT1 proteins simultaneously using a gold-coated sensor chip.In contrast to conventional techniques, which mainly operate with voltage steps, currents are triggered by sugar or sodium addition. Sugar concentration jumps in the presence of sodium lead to transport currents between 5 and 10 nA. Remarkably, in the absence of sodium (i.e. no transport), we observed fast pre-steady-state (PSS) currents with time constants between 3 and 10 ms. These PSS currents mainly originate from sugar binding. Sodium binding does not induce PSS currents. Due to high time resolution, PSS currents were distinguished from transport and eventually correlated with conformational transitions within the sugar translocation pathway.In addition, we analyzed the impact of driving forces on transport and binding currents, showing that membrane voltage and sodium concentration gradients lead to an increased transport rate without affecting sugar binding kinetics. We also compared Na+/sugar efflux with physiologically relevant influx and found similar transport rates, but lower affinity in efflux mode.SSM-based electrophysiology is a powerful technique, which overcomes bottlenecks for transport measurements observed in other techniques such as the requirement of labels or the lack of real-time data. Rapid solution exchange enables the observation of substrate-induced electrogenic events like conformational transitions, opening novel perspectives for in-depth functional studies of SGLT1 and other transporters.

Classic Pentachlorophenol Hydroxylating Phenylalanine 4-Monooxygenase from Indigenous Bacillus tropicus Strain AOA-CPS1: Cloning, Overexpression, Purification, Characterization and Structural Homology Modelling.

The metabolically promiscuous pentachlorophenol (PCP) hydroxylating Phe4MO (represented as CpsB) was detected, amplified (from the genome of Bacillus tropicus strain AOA-CPS1), cloned, overexpressed, purified and characterized here. The 1.755-kb gene cloned in the pET15b vector expressed a ≅ 64kDa monomeric protein which was purified to homogeneity by single-step affinity chromatography, with a total yield of 82.1%. The optimum temperature and pH of the enzyme were found to be 30°C and 7.0, respectively. CpsB showed functional stability between pH 6.0-7.5 and temperature 25-30°C. The enzyme-substrate reaction kinetic studies showed the allosteric nature of the enzyme and followed pre-steady state using NADH as a co-substrate with apparent vmax, Km, kcat and kcat/Km values of 0.465μM.s-1, 140μM, 0.099s-1 and 7.07 × 10-4μM-1.s-1, respectively, for the substrate PCP. The in-gel trypsin digestion experiments and bioinformatic tools confirmed that the reported enzyme is a Phe4MO with multiple putative conserved domains and metal ion-binding site. Though Phe4MO has been reported to have a diverse catalytic function, here we report, for the first time, that it functions as a PCP dehalogenase or PCP-4-monooxygenase by hydroxylating PCP. Hence, the use of this enzyme may be further explored in the bioremediation of PCP and other related xenobiotics.

A guide to the Michaelis-Menten equation: steady state and beyond.

The modern definition of enzymology is synonymous with the Michaelis-Menten equationinstituted by Leonor Michaelis and Maud Menten. Most textbooks, or chapters within, discussing enzymology start with the derivation of the equationunder the assumption of rapid equilibrium (as done by Michaelis-Menten) or steady state (as modified by Briggs and Haldane) conditions to highlight the importance of this equationas the bedrock on which interpretation of enzyme kinetic results is dependent. However, few textbooks or monographs take the effort of placing the equationwithin its right historical context and discuss the assumptions that have gone into its institution. This guide will dwell on these in substantial detail. Further, this guide will attempt to instil a sense of appreciation for the mathematical curve rectangular hyperbola, its unique attributes and how ubiquitous the curve is in biological systems. To conclude, this guide will discuss the limitations of the equation, and the method it embodies, and trace the journey of how investigators are attempting to move beyond the steady-state approach and the Michaelis-Menten equationinto full progress curve, pre-steady state and single-turnover kinetic analysis to obtain greater insights into enzyme kinetics and catalysis.

L-DOPA Dioxygenase Activity on 6-Substituted Dopamine Analogues.

Dioxygenase enzymes are essential protein catalysts for the breakdown of catecholic rings, structural components of plant woody tissue. This powerful chemistry is used in nature to make antibiotics and other bioactive materials or degrade plant material, but we have a limited understanding of the breadth and depth of substrate space for these potent catalysts. Here we report steady-state and pre-steady-state kinetic analysis of dopamine derivatives substituted at the 6-position as substrates of L-DOPA dioxygenase, and an analysis of that activity as a function of the electron-withdrawing nature of the substituent. Steady-state and pre-steady-state kinetic data demonstrate the dopamines are impaired in binding and catalysis with respect to the cosubstrate molecular oxygen, which likely afforded spectroscopic observation of an early reaction intermediate, the semiquinone of dopamine. The reaction pathway of dopamine in the pre-steady state is consistent with a nonproductive mode of binding of oxygen at the active site. Despite these limitations, L-DOPA dioxygenase is capable of binding all of the dopamine derivatives and catalyzing multiple turnovers of ring cleavage for dopamine, 6-bromodopamine, 6-carboxydopamine, and 6-cyanodopamine. 6-Nitrodopamine was a single-turnover substrate. The variety of substrates accepted by the enzyme is consistent with an interplay of factors, including the capacity of the active site to bind large, negatively charged groups at the 6-position and the overall oxidizability of each catecholamine, and is indicative of the utility of extradiol cleavage in semisynthetic and bioremediation applications.

The dynamic cycle of bacterial translation initiation factor IF3.

Initiation factor IF3 is an essential protein that enhances the fidelity and speed of bacterial mRNA translation initiation. Here, we describe the dynamic interplay between IF3 domains and their alternative binding sites using pre-steady state kinetics combined with molecular modelling of available structures of initiation complexes. Our results show that IF3 accommodates its domains at velocities ranging over two orders of magnitude, responding to the binding of each 30S ligand. IF1 and IF2 promote IF3 compaction and the movement of the C-terminal domain (IF3C) towards the P site. Concomitantly, the N-terminal domain (IF3N) creates a pocket ready to accept the initiator tRNA. Selection of the initiator tRNA is accompanied by a transient accommodation of IF3N towards the 30S platform. Decoding of the mRNA start codon displaces IF3C away from the P site and rate limits translation initiation. 70S initiation complex formation brings IF3 domains in close proximity to each other prior to dissociation and recycling of the factor for a new round of translation initiation. Altogether, our results describe the kinetic spectrum of IF3 movements and highlight functional transitions of the factor that ensure accurate mRNA translation initiation.

P010. Evaluation of the Efficacy and Safety of AKCEA-TTR-LRx (ION-682884) in Patients with Transthyretin-mediated Amyloid Cardiomyopathy: The CARDIO-TTRansform Study

<h3>Background</h3> Transthyretin amyloidosis cardiomyopathy (ATTR-CM) is a fatal condition, leading to heart failure (HF) and ultimately death. ATTR-CM is caused by misfolding and aggregation of transthyretin (TTR), a protein produced by the liver. Depending on the presence or absence of a destabilizing mutation in the TTR gene, the disease can be classified as hereditary ATTR-CM (hATTR-CM) or wild-type ATTR-CM (wtATTR-CM), respectively. Despite the treatment with a TTR stabilizer, tafamidis, recently approved in the United States for the treatment of ATTR-CM, disease progression still occurs. AKCEA-TTR-L_RX (ION-682884) is an antisense oligonucleotide (ASO) that inhibits the production of TTR. It has a similar design and sequence as inotersen (the parent compound), but is conjugated to a triantennary N-acetyl galactosamine (GalNAc3) moiety for selective receptor-mediated delivery to hepatocytes, the principal source of systemically circulating TTR. This delivery approach has yielded an up to 30-fold increase in potency and improved the safety and tolerability profiles of ASOs in human clinical trials. Conjugation of this ligand allows the use of a lower dose to achieve improved pharmacodynamic results. In a phase 1, randomized, placebo-controlled study, AKCEA-TTR-L_RX given at a 45 mg, 60 mg or 90 mg dose, by subcutaneous (SC) injection every four weeks in 36 healthy volunteers achieved a mean pre-steady state reduction in serum TTR of 86%, 91% and 94%, respectively, compared to baseline. The dosage regimen of 45 mg SC every four weeks (27-fold lower exposure vs the inotersen dose in NEURO-TTR trial) was chosen for the pivotal phase 3 study. <h3>Methods</h3> CARDIO-TTRansform (ClinicalTrials.gov NCT04136171) is a Phase 3 global, double-blind, randomized, placebo-controlled study assessing the efficacy and safety of AKCEA-TTR-L _Rx in hATTR-CM or wtATTR-CM patients receiving available background standard of care (SoC) therapy. Approximately 750 patients around the world with a history of HF due to ATTR-CM will be randomized 1:1 to receive either AKCEA-TTR-L_Rx 45 mg or placebo administered by SC injection once every 4 weeks. Key inclusion criteria include diagnosis of ATTR-CM by biopsy or positive PYP/DPD/HMDP scan, interventricular septum thickness >12mm, NT-proBNP > 600 pg/mL, NYHA class I-III and 6-minute walk distance (6MWD) >150 m. Key exclusion criteria include, platelet count < 125 × 10⁹\L and urine protein/creatinine ratio >= 750 mg/g. Concomitant treatment with tafamidis as SoC for ATTR-CM is allowed. The study consists of a 120-week Treatment Period. Primary efficacy endpoint is the composite of cardiovascular (CV) mortality and recurrent CV clinical events at Week 120 study visit using the Andersen-Gill method. Secondary endpoints include the change from baseline in the 6MWD, KCCQ score, CV clinical events, CV death and all-cause of mortality at Week 120. <h3>Conclusions</h3> Despite recent advances, additional efficacious, safe and convenient treatment options for ATTR-CM are needed. The CARDIO-TTRansform trial is a large Phase 3 trial designed to evaluate the clinical efficacy and safety of AKCEA-TTR-LRx compared to placebo in patients with ATTR-CM receiving available SoC therapy.

Immune phase transition under steroid treatment

The steroid hormone glucocorticoid (GC) is a well-known immunosuppressant that controls T-cell-mediated adaptive immune response. In this work, we have developed a minimal kinetic network model of T-cell regulation connecting relevant experimental and clinical studies to quantitatively understand the long-term effects of GC on pro-inflammatory T-cell (T_{pro}) and anti-inflammatory T-cell (T_{anti}) dynamics. Due to the antagonistic relation between these two types of T cells, their long-term steady-state population ratio helps us to characterize three classified immune regulations: (i) weak ([T_{pro}]>[T_{anti}]), (ii) strong ([T_{pro}]<[T_{anti}]), and (iii) moderate ([T_{pro}]∼[T_{anti}]), holding the characteristic bistability. In addition to the differences in their long-term steady-state outcome, each immune regulation shows distinct dynamical phases. In the presteady state, a characteristic intermediate stationary phase is observed to develop only in the moderate regulation regime. In the medicinal field, the resting time in this stationary phase is distinguished as a clinical latent period. GC dose-dependent steady-state analysis shows an optimal level of GC to drive a phase transition from the weak or autoimmune prone to the moderate regulation regime. Subsequently, the presteady state clinical latent period tends to diverge near that optimal GC level where [T_{pro}]:[T_{anti}] is highly balanced. The GC-optimized elongated stationary phase explains the rationale behind the requirement of long-term immune diagnostics, especially when long-term GC-based chemotherapeutics and other immunosuppressive drugs are administrated. Moreover, our study reveals GC sensitivity of clinical latent period, which might serve as an early warning signal in diagnosing different immune phases and determining immune phasewise steroid treatment.

Kinetic Analysis Suggests Evolution of Ribosome Specificity in Modern Elongation Factor-Tus from "Generalist" Ancestors.

It has been hypothesized that early enzymes are more promiscuous than their extant orthologs. Whether or not this hypothesis applies to the translation machinery, the oldest molecular machine of life, is not known. Efficient protein synthesis relies on a cascade of specific interactions between the ribosome and the translation factors. Here, using elongation factor-Tu (EF-Tu) as a model system, we have explored the evolution of ribosome specificity in translation factors. Employing presteady state fast kinetics using quench flow, we have quantitatively characterized the specificity of two sequence-reconstructed 1.3- to 3.3-Gy-old ancestral EF-Tus toward two unrelated bacterial ribosomes, mesophilic Escherichia coli and thermophilic Thermus thermophilus. Although the modern EF-Tus show clear preference for their respective ribosomes, the ancestral EF-Tus show similar specificity for diverse ribosomes. In addition, despite increase in the catalytic activity with temperature, the ribosome specificity of the thermophilic EF-Tus remains virtually unchanged. Our kinetic analysis thus suggests that EF-Tu proteins likely evolved from the catalytically promiscuous, “generalist” ancestors. Furthermore, compatibility of diverse ribosomes with the modern and ancestral EF-Tus suggests that the ribosomal core probably evolved before the diversification of the EF-Tus. This study thus provides important insights regarding the evolution of modern translation machinery.

The RED scheme: Rate-constant estimation from pre-steady state weighted ensemble simulations.

We present the Rate from Event Durations (RED) scheme, a new scheme that more efficiently calculates rate constants using the weighted ensemble path sampling strategy. This scheme enables rate-constant estimation from shorter trajectories by incorporating the probability distribution of event durations, or barrier-crossing times, from a simulation. We have applied the RED scheme to weighted ensemble simulations of a variety of rare-event processes that range in complexity: residue-level simulations of protein conformational switching, atomistic simulations of Na+/Cl- association in explicit solvent, and atomistic simulations of protein-protein association in explicit solvent. Rate constants were estimated with up to 50% greater efficiency than the original weighted ensemble scheme. Importantly, our scheme accounts for the systematic error that results from statistical bias toward the observation of events with short durations and reweights the event duration distribution accordingly. The RED scheme is relevant to any simulation strategy that involves unbiased trajectories of similar length to the most probable event duration, including weighted ensemble, milestoning, and standard simulations as well as the construction of Markov state models.

LIG1 syndrome mutations remodel a cooperative network of ligand binding interactions to compromise ligation efficiency.

Human DNA ligase I (LIG1) is the main replicative ligase and it also seals DNA breaks to complete DNA repair and recombination pathways. Immune compromised patients harbor hypomorphic LIG1 alleles encoding substitutions of conserved arginine residues, R771W and R641L, that compromise LIG1 activity through poorly defined mechanisms. To understand the molecular basis of LIG1 syndrome mutations, we determined high resolution X-ray structures and performed systematic biochemical characterization of LIG1 mutants using steady-state and pre-steady state kinetic approaches. Our results unveil a cooperative network of plastic DNA-LIG1 interactions that connect DNA substrate engagement with productive binding of Mg2+ cofactors for catalysis. LIG1 syndrome mutations destabilize this network, compromising Mg2+ binding affinity, decreasing ligation efficiency, and leading to elevated abortive ligation that may underlie the disease pathology. These findings provide novel insights into the fundamental mechanism by which DNA ligases engage with a nicked DNA substrate, and they suggest that disease pathology of LIG1 syndrome could be modulated by Mg2+ levels.

Interactions of a Signal Transduction Protein Investigated by Fluorescence Stopped-Flow Kinetics.

To understand cellular processes such as biochemical pathways and signaling networks, we need to understand binding and reaction rates of often competing reactions, their dependence on cellular concentrations of participating molecules, and the regulation of these rates through allostery, posttranslational modifications, or other mechanisms. To do so, we break these systems down into their elementary steps, which are almost invariably either unimolecular or bimolecular reactions that frequently occur on sub-second, often sub-millisecond, time scales. Rapid mixing techniques, which generally achieve mixing in less than 2ms, are generally suitable for the study of such reactions. The application of these techniques to the study of enzyme mechanisms is described in several excellent texts (Cornish-Bowden, Fundamentals of enzyme kinetics, 1995; Gutfreund, Kinetics for the life sciences. Receptors, transmitters and catalysis, 1995); flow techniques are used to study individual steps by monitoring the approach to equilibrium (the pre-steady state) under single turnover conditions.The individual steps in complex biochemical reaction schemes determine how fast systems can respond to incoming signals and adapt to changed conditions[1, 2]. This chapter is concerned with in vitro techniques that have been developed to study fast reactions in solution, and we present the study of various interactions of calmodulin as an example. The kinetic information obtained with these techniques is indispensable for understanding the dynamics of biochemical processes and complements the static structural and thermodynamic information available from X-ray crystallography, NMR, and equilibrium binding studies.

Measurement of Nucleotide Hydrolysis Using Fluorescent Biosensors for Phosphate.

Assays for the detection of inorganic phosphate (Pi) are widely used to measure the activity of nucleotide hydrolyzing enzymes, such as ATPases and GTPases. The fluorescent biosensors for Pi, described here, are based on fluorescently labeled versions of E. coli phosphate-binding protein (PBP), which translates Pi binding into a large change in fluorescence intensity. In comparison with other Pi-detection systems, these biosensors are characterized by a high sensitivity (sub-micromolar Pi concentrations) and high time resolution (tens of milliseconds), and they are therefore particularly well suited for measurements of phosphate ester hydrolysis in real time. In this chapter, it is described how the Pi biosensors can be used to measure kinetics of ATPase and GTPase reactions, both under steady state and pre-steady state conditions. An example protocol is given for determining steady state kinetic parameters, Km and kcat, of the ATP-dependent chromatin remodeler Chd1, in a plate reader format. In addition, the measurement of Pi release kinetics under pre-steady state conditions is described, including a detailed experimental procedure for a singleturnover measurement of ATP hydrolysis by the ABC-type ATPase SufBC using rapid mixing.

A Quick Primer in Fluorescence-Based Equilibrium and Pre-steady State Methods for Determining Protein-Nucleotide Affinities.

Biomolecular interactions facilitate the biochemical processes that sustain life. Proteins, RNAs, and ribonucleoprotein complexes perform cellular functions that range from catalyzing the formation or cleavage of bonds to being structural building blocks, both of which are only possible through the interaction with their respective biomolecular partner(s). Having access to the parameters that describe these interactions is important for our understanding of the principles that underlie enzymatic and nonenzymatic processes. Here we describe two fluorescence-based approaches to determine two key parameters, the affinity and the rate of association/dissociation of a protein and a ligand. Considerations are provided to expand the described approach to other experimental systems.

Kinetic Analysis of Transient Intermediates in the Mechanism of Prenyl-Flavin-Dependent Ferulic Acid Decarboxylase.

Ferulic acid decarboxylase catalyzes the decarboxylation of various substituted phenylacrylic acids to their corresponding styrene derivatives and CO2 using the recently discovered cofactor prenylated FMN (prFMN). The mechanism involves an unusual 1,3-dipolar cycloaddition reaction between prFMN and the substrate to generate a cycloadduct capable of undergoing decarboxylation. Using native mass spectrometry, we show the enzyme forms a stable prFMN-styrene cycloadduct that accumulates on the enzyme during turnover. Pre-steady state kinetic analysis of the reaction using ultraviolet-visible stopped-flow spectroscopy reveals a complex pattern of kinetic behavior, best described by a half-of-sites model involving negative cooperativity between the two subunits of the dimeric enzyme. For the reactive site, the cycloadduct of prFMN with phenylacylic acid is formed with a kapp of 131 s-1. This intermediate converts to the prFMN-styrene cycloadduct with a kapp of 75 s-1. Cycloelimination of the prFMN-styrene cycloadduct to generate styrene and free enzyme appears to determine kcat for the overall reaction, which is 11.3 s-1.

Characterization of the Kinetic Mechanism of Human Protein Arginine Methyltransferase 5.

Protein arginine methyltransferases (PRMTs) are of great interest for the development of therapeutics due to their involvement in a number of malignancies, such as lung and colon cancer. PRMT5 catalyzes the formation of symmetrical dimethylarginine of a wide variety of substrates and is responsible for the majority of this mark within cells. To gain insight into the mechanism of PRMT5 inhibition, we co-expressed the human PRMT5:MEP50 complex (hPRMT5:MEP50) in insect cells for a detailed mechanistic study. In this report, we carry out steady state, product, and dead-end inhibitor studies that show hPRMT5:MEP50 uses a rapid equilibrium random order mechanism with EAP and EBQ dead-end complexes. We also provide evidence of ternary complex formation in solution using hydrogen/deuterium exchange mass spectrometry. Isotope exchange and intact protein mass spectrometry further rule out ping-pong as a potential enzyme mechanism, and finally, we show that PRMT5 exhibits a pre-steady state burst that corresponds to an initial slow turnover with all four active sites of the hetero-octamer being catalytically active.

Real-time investigation of the roles of ATP hydrolysis by UvrA and UvrB during DNA damage recognition in nucleotide excision repair

Nucleotide excision repair (NER) stands out among other DNA repair systems for its ability to process a diverse set of unrelated DNA lesions. In bacteria, NER damage detection is orchestrated by the UvrA and UvrB proteins, which form the UvrA2-UvrB2 (UvrAB) damage sensing complex. The highly versatile damage recognition is accomplished in two ATP-dependent steps. In the first step, the UvrAB complex samples the DNA in search of lesion. Subsequently, the presence of DNA damage is verified within the UvrB-DNA complex after UvrA has dissociated. Although the mechanism of bacterial NER damage detection has been extensively investigated, the role of ATP binding and hydrolysis by UvrA and UvrB during this process remains incompletely understood. Here, we report a pre-steady state kinetics Förster resonance energy transfer (FRET) study of the real-time interaction between UvrA, UvrB, and damaged DNA during lesion detection. By using UvrA and UvrB mutants harboring site-specific mutations in the ATP binding sites, we show for the first time that the dissociation of UvrA from the UvrAB-DNA complex does not require ATP hydrolysis by UvrB. We find that ATP hydrolysis by UvrA is not essential, but somehow facilitates the formation of UvrB-DNA complex, with ATP hydrolysis at the proximal site of UvrA playing a more critical role. Consistent with previous reports, our results indicated that the ATPase activity of UvrB is essential for the formation of UvrB-DNA complex but is not required for the binding of the UvrAB complex to DNA.

S224 Presents a Catalytic Trade-off in PLP-Dependent l-Lanthionine Synthase from Fusobacterium nucleatum.

Lanthionine synthase from the oral bacterium Fusobacterium nucleatum is a fold type II pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the β-replacement of l-cysteine by a second molecule of l-cysteine to form H2S and l-lanthionine. The meso-isomer of the latter product is incorporated into the F. nucleatum peptidoglycan layer. Herein, we investigated the catalytic role of S224, which engages in hydrogen-bond contact with the terminal carboxylate of l-lanthionine in the closed conformation of the enzyme. Unexpectedly, the S224A variant elicited a 7-fold increase in the turnover rate for H2S and lanthionine formation and a 70-fold faster rate constant for the formation of the α-aminoacrylate intermediate compared to the wild-type enzyme. Presteady state kinetic analysis further showed that the reaction between S224A and l-cysteine leads to the formation of the more reactive ketoenamine tautomer of the α-aminoacrylate. The α-aminoacrylate with the protonated Schiff base is not an observable intermediate in the analogous reaction with the wild type, which may account for its attenuated kinetic properties. However, the S224A substitution is detrimental to other aspects of the catalytic cycle; it facilitates the α,β-elimination of l-lanthionine, and it weakens the enzyme's catalytic preference for the formation of l-lanthionine over that of l-cystathionine.

Temperature controlled high-throughput magnetic tweezers show striking difference in activation energies of replicating viral RNA-dependent RNA polymerases.

RNA virus survival depends on efficient viral genome replication, which is performed by the viral RNA dependent RNA polymerase (RdRp). The recent development of high throughput magnetic tweezers has enabled the simultaneous observation of dozens of viral RdRp elongation traces on kilobases long templates, and this has shown that RdRp nucleotide addition kinetics is stochastically interrupted by rare pauses of 1–1000 s duration, of which the short-lived ones (1–10 s) are the temporal signature of a low fidelity catalytic pathway. We present a simple and precise temperature controlled system for magnetic tweezers to characterize the replication kinetics temperature dependence between 25°C and 45°C of RdRps from three RNA viruses, i.e. the double-stranded RNA bacteriophage Φ6, and the positive-sense single-stranded RNA poliovirus (PV) and human rhinovirus C (HRV-C). We found that Φ6 RdRp is largely temperature insensitive, while PV and HRV-C RdRps replication kinetics are activated by temperature. Furthermore, the activation energies we measured for PV RdRp catalytic state corroborate previous estimations from ensemble pre-steady state kinetic studies, further confirming the catalytic origin of the short pauses and their link to temperature independent RdRp fidelity. This work will enable future temperature controlled study of biomolecular complex at the single molecule level.

Pre‐Steady State Kinetic Analysis of DnaE Polymerase Illuminates the Limited (But Essential) Role It Performs in Gram‐Positive DNA Replication

PolC and DnaE are the essential C‐family replicative polymerases in the Gram‐positive pathogen Staphylococcus aureus. Both PolC and DnaE have fast catalytic rates on DNA (PolC kpol, 500–1000 s−1; DnaE kpol, 300–400 s−1) however, we know PolC performs the bulk of genome replication. PolC is unable to initiate synthesis from RNA primers and relies on DnaE to perform this role before taking over replication. What remains to be determined is where and how this polymerase switching occurs. Using rapid quench‐flow we have performed detailed pre‐steady state kinetic analysis of DnaE on both a DNA‐ and RNA‐primed substrate. We have determined key kinetic properties that explain why DnaE has been confined to its role of replication initiation. DnaE shows a comparable affinity for both DNA‐ and RNA‐primed templates (Kd: 7 nM vs. 18 nM), but DnaE falls off an RNA‐primed template 50‐fold faster than a DNA‐primed template (koff: 5.34 s−1 vs. 0.11 s−1). Additionally, when incorporating a nucleotide on an RNA‐primed template, DnaE demonstrates a slower catalytic rate (kpol: 120 s−1), reversible chemistry, and a rate‐limiting pyrophosphate release rate (kPPi: ~20 s−1), which is not observed on a DNA‐primed template. These critical kinetic characteristics explain the limited role DnaE plays in genome replication. When extending from an RNA‐primed substrate DnaE is slow and falls off the substrate rapidly after adding a few nucleotides. This provides a critical juncture for PolC to compete for binding and finish replicating the template.Support or Funding InformationNational Institute of General Medical SciencesProject Number: 5R01GM080573‐09