The identification of chemical intermediates in enzyme catalysis by the rapid quench-flow technique

Barman, Tom; Bellamy, Stuart R.W.; Gutfreund, H.; Halford, Stephen E.; Lionne, Corinne

doi:10.1007/s00018-006-6243-z

Cited by 36 publications

(22 citation statements)

References 74 publications

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“…More investigations are necessary to distinguish among the three chemical reactions in pseudouridine catalysis and to identify the rate-limiting step within these reactions. Importantly, the quench-flow technique used here might help to isolate and characterize transient intermediates on the reaction pathway (Barman et al 2006). Interestingly, the first substep, cleavage of the N-glycosidic bond, resembles the reaction catalyzed by uracil-DNA glycosylases, which display k cat values of 4-200 sec À1 (Duraffour et al 2007;Liu et al 2007), at least 10-fold higher than the catalytic rate constant of pseudouridine formation reported here.…”

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confidence: 78%

Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis

Wright

Keffer-Wilkes²,

Dobing³

et al. 2011

RNA

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Pseudouridine synthases catalyze formation of the most abundant modification of functional RNAs by site-specifically isomerizing uridines to pseudouridines. While the structure and substrate specificity of these enzymes have been studied in detail, the kinetic and the catalytic mechanism of pseudouridine synthases remain unknown. Here, the first pre-steady-state kinetic analysis of three Escherichia coli pseudouridine synthases is presented. A novel stopped-flow absorbance assay revealed that substrate tRNA binding by TruB takes place in two steps with an overall rate of 6 sec À1 . In order to observe catalysis of pseudouridine formation directly, the traditional tritium release assay was adapted for the quench-flow technique, allowing, for the first time, observation of a single round of pseudouridine formation. Thereby, the single-round rate constant of pseudouridylation (k C ) by TruB was determined to be 0.5 sec À1 . This rate constant is similar to the k cat obtained under multiple-turnover conditions in steady-state experiments, indicating that catalysis is the rate-limiting step for TruB. In order to investigate if pseudouridine synthases are characterized by slow catalysis in general, the rapid kinetic quench-flow analysis was also performed with two other E. coli enzymes, RluA and TruA, which displayed rate constants of pseudouridine formation of 0.7 and 0.35 sec À1 , respectively. Hence, uniformly slow catalysis might be a general feature of pseudouridine synthases that share a conserved catalytic domain and supposedly use the same catalytic mechanism.

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confidence: 78%

Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis

Wright

Keffer-Wilkes²,

Dobing³

et al. 2011

RNA

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“…Two techniques have been used to rapidly change the concentration of compounds in myofibrils: (1) rapid mixing of myofibril suspensions with buffers in a reaction chamber by stopped flow or quench flow to study the transient kinetics of myofibril ATPase and myofibril Ca 2+ regulation [7,8,58,64,88,92,98,132,134] and (2) rapid switching between two laminar flows of solution ejected by a double-channel micropipette to induce force kinetics and sarcomere dynamics in single myofibrils mounted in a force-recording apparatus [26]. The principle of this technique is illustrated in Fig.…”

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confidence: 99%

Insights into the kinetics of Ca2+-regulated contraction and relaxation from myofibril studies

Stehle

Solzin

Iorga

et al. 2009

Pflugers Arch - Eur J Physiol

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Muscle contraction results from force-generating interactions between myosin cross-bridges on the thick filament and actin on the thin filament. The force-generating interactions are regulated by Ca(2+) via specialised proteins of the thin filament. It is controversial how the contractile and regulatory systems dynamically interact to determine the time course of muscle contraction and relaxation. Whereas kinetics of Ca(2+)-induced thin-filament regulation is often investigated with isolated proteins, force kinetics is usually studied in muscle fibres. The gap between studies on isolated proteins and structured fibres is now bridged by recent techniques that analyse the chemical and mechanical kinetics of small components of a muscle fibre, subcellular myofibrils isolated from skeletal and cardiac muscle. Formed of serially arranged repeating units called sarcomeres, myofibrils have a complete fully structured ensemble of contractile and Ca(2+) regulatory proteins. The small diameter of myofibrils (few micrometres) facilitates analysis of the kinetics of sarcomere contraction and relaxation induced by rapid changes of [ATP] or [Ca(2+)]. Among the processes studied on myofibrils are: (1) the Ca(2+)-regulated switch on/off of the troponin complex, (2) the chemical steps in the cross-bridge adenosine triphosphatase cycle, (3) the mechanics of force generation and (4) the length dynamics of individual sarcomeres. These studies give new insights into the kinetics of thin-filament regulation and of cross-bridge turnover, how cross-bridges transform chemical energy into mechanical work, and suggest that the cross-bridge ensembles of each half-sarcomere cooperate with each other across the half-sarcomere borders. Additionally, we now have a better understanding of muscle relaxation and its impairment in certain muscle diseases.

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“…Quenched-flow is a similar technique to stopped-flow, whereby two solutions can be rapidly mixed with one another, followed by a second mixing with a quench agent (Fig. 4.4a) [22,23]. This is typically an acid to denature the ATPase, which therefore stops the reaction at chosen time points [4].…”

Section: Hydrolysis Stepmentioning

confidence: 99%

Fluorescence to Study the ATPase Mechanism of Motor Proteins

Toseland

2014

Experientia Supplementum

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This chapter provides an overview of different methodologies to dissect the ATPase mechanism of motor proteins. The use of ATP is fundamental to how these molecular engines work and how they can use the energy to perform various cellular roles. Rapid reaction and single-molecule techniques will be discussed to monitor reactions in real time through the application of fluorescence intensity, anisotropy and FRET. These approaches utilise fluorescent nucleotides and biosensors. While not every technique may be suitable for your motor protein, the different ways to determine the ATPase mechanism should allow a good evaluation of the kinetic parameters.

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The identification of chemical intermediates in enzyme catalysis by the rapid quench-flow technique

Cited by 36 publications

References 74 publications

Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis

Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis

Insights into the kinetics of Ca2+-regulated contraction and relaxation from myofibril studies

Fluorescence to Study the ATPase Mechanism of Motor Proteins

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