HESIOD THEOG SOLON
HESIOD THEOG SOLON GREEK 161
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Cell Mol Life Sci 63 2006 257172583 1420682X0622257113 DOI 101007s000180066243z Birkhauser Verlag Basel 2006 Review lCellular and Molecular Life Sciences The identification of chemical intermediates in enzyme catalysis by the rapid quench ow technique T E Barman 3 S R W Bellamyquot H Gutfreund b S E Halfordquot and C Lionne 3 7 UMR 5121 CNRSUniversity Montpellier 1 Institut de Biologie 4 bd Henri 1V 34000 Montpellier France Fax 33 467 604 420 email corinnelionneunivmontplfr b Department of Biochemistry School of Medical Sciences University of Bristol Bristol BS8 lTD United Kingdom Received 24 May 2006 received after revision 3 July 2006 accepted 19 July 2006 Online First 4 September 2006 Abstract Traditionally enzyme transient kinetics have been studied by the stopped ow and rapid quench ow QF methods Whereas stopped ow is the more con venient it suffers from two weaknesses optically silent systems cannot be studied and when there is a signal it cannot always be assigned to a particular step in the reac tion pathway QF is a chemical sampling method reac tion mixtures are aged for a few milliseconds or longer stopped by a quenching agent and the product or the intermediate is measured by a specific analytical method Here we show that by exploiting the array of current ana lytical methods and different quenching agents the QF method is a key technique for identifying and for char acterising kinetically intermediates in enzyme reaction pathways and for determining the order by which bonds are formed or cleaved by enzymes acting on polymer sub strates such as DNA Keywords Enzyme mechanism protein dynamics rapidreaction technique kinetics ATPase DNAprotein inter action Introduction The study of transient kinetics of enzyme reactions ne cessitates the observation of intermediate and product formation during the short time course from mixing en zyme and substrate until the steady state or equilibrium is reached There are two complementary approaches to the application of transient kinetics to the exploration of mechanisms What might be called its grammar requires the algebraic resolution of the exponentials time con stants and amplitudes describing the time course of the transient concentration changes The principles of various methods pertinent to this approach have been described in a recent book 1 The secondpart ofthe study oftran sient kinetics is the optical andor chemical identification of the intermediates that occur on the timescales of tran sients It is worthwhile reemphasising the complemen Corresponding author tarity of the two approaches Here we review the contri butions made by the quench ow QF technique to the identification and determination of lifetimes of chemical intermediates during transients QF first receivedlirnited attention 70 years ago fromF J W Roughton the father of rapid ow techniques for the study of reactions of C02 2 Its time resolution has since been improved by an order of magnitude to about 3 ms 3 The relatively limited use of QF despite its improvements and application to enzymecatalysed reactions is due to a number of factors First and foremost biochemists have become used to applying methods that give a continu ous record of the reaction regardless of whether they are studying steadystate or transient kinetics The QF method entails the individual analysis of each time sample and therefore requires more time and effort However steady improvements have been made to QF equipment in recent years to economise in reactant volumes and to simplify sampling Furthermore modern analytical techniques 2572 T E Barman Ct 211 have vastly extended the mnge and ease of applications f 39 39 39 39 39 reactio The quenchr ow method In this review We use selected examples to demonstrate T GL 4 in biochemistry thought that QF developed by Barman amp Gutfreund 3 Was an interesting method but not likely to find Wide application This Was Written at a time 1966 rates of cleavage steps in the reactions of trypsin chymotrypsin and alkaline phosphatase 5 6 In subsequent years many fruitful applications largely though by no means entirely 39th reactions involving phosphate compounds have dem l L L of L L L istry of What Britton Chance called kinetically competent intermediates and placing them in a sequence ont e pat Way ofenzymecatalysed reactions We do not intend to 391 L l 4 LL L L L by this method The examples discussed have been se lected to highlight either specific technical applications or semin contributions to biochemical mec anisms We hope to demonstrate that the more laborious procedure of LL L troscopically silent intermediates and their chemistry 7 This asmadet etechnique su iciently popularto justify the production of four commercial instruments WWW tgkscientificcom i 39 39 f 39 WWWphotophysicscom More surprising is the frequent underestimation of the capacity of the technique for development or adjust ments making it possible to use QF to dissect a Wide al example is the release 8 This last step is the one that is the most dif L peating the mixing of the reactants many times and stop g the progress of the reaction after Welldefined time intervals allows the accurate chemical identification of E Experimental All ow techniques depend on e icient mixing of reac tants Apart from some very elaborate procedures mix ing times of 1 ms have not been significantly improve i u 1 1 ing techniques are likely to disrupt the noncovalent uct complexes that precede the final dis sociation of the end product This is connected to the zyme reactions unlike classical Mi chaelisMenten models the chemistry is not rate limit during turnover that can be identified chemical For example We Will describe below hoW on enzyme equilibria i e ennililm39a k t t 1 enzymeproduct complexes have been characterised in ATPases by this technique Enzyme Substrate capillary me mmng chamber Quencher since the f39 of oW methods 80 years ago The QF technique depends on asecond mixing event the reaction mixture With a quenching reagent at variable 7 but defined 7 time intervals after the first The equipment The QF technique is a chemical sampling method This is a stande method trate mi Whilst samples are re experiments are carried out manually overtime sc es of tens of seconds to minutes They are thus usually limited quenched sample collection tube 1 39 y 439 A Janquot om mum the capillary tube tube B wryingV L quotm r and thequot tnn 39 ML Tquot mama Cell Mol Life Sci Vol 63 2006 to obtaining overall time courses of product formation at low enzyme concentrations To detect intermediates chemical sampling has to be carried out at high enzyme concentrations in the millisecond time range and there fore requires fast reaction equipment such as QF Early QF apparatuses consisted simply of two syringes that contained the reagents connected to a mixer and a capillary tube dipped in the quenching solution as in Figure la Typically the syringes were driven by a vari able speed motor via a clutch and brake unit operated by switches 3 This simple method has two limitations First experiments are limited to a maximum time of about 300 ms For longer times slow ow rates result in poor mixing Second it is wasteful because it requires large volumes of reaction mixture 7 typically 0571 ml per push To overcome these limitations two important technical improvements have been made First to reduce the large volumes needed Eccleston et al 10 constructed a lowvolume QF apparatus in which the reactants are loaded into loops between the mixing chamber and drive syringes which contain buf fer only For an experiment the driving syringes are ac tivated the buffer pushes the reagents into the mixing chamber and the slug of reaction mixture is quenched in a second mixer and then analysed With this type of apparatus reaction mixtures can be reduced to 50 ul or even less Second to allow for reaction times gt 300 ms Fersht and lakes 1 l constructed atimedelay or pulseflow QF ap paratus The principle of this is illustrated in Figure lb In commercial QF equipment such as those from TgK Scientific BioLogic or KinTek the reaction mixtures are less than 100 ul and the continuousflow and timede lay modes are combined Further the syringes are driven by stepping motors that are controlled by computers features that facilitate greatly the operators work Alter natively Applied Photophysics provide a QF adaptor for their stoppedflow apparatus Experimental strategies The following aspects of experimental design must be considered First what is to be assayed Ideally one would like to obtain the concentration of each interme diate on a reaction pathway but in actual practice this is usually reduced to measuring a product For example when reaction mixtures are quenched in strong acid it is assumed that any enzyme intermediate with bound prod uct decomposes so the measurement reports total rather than just free product Thus when product release is slow one can obtain the kinetics of formation of enzyme product intermediates as well as those of the release of products Information on different intermediates can be obtained by changing the quenching agent indeed an important Review Article 2573 feature of the QF method is that one can stop reactions in different quenching media For example when reac tions of myosin with y 3ZPATP are quenched with un labelledATP the kinetics of the ATPbinding process are obtained specifically whereas when they are quenched in acid the kinetics of ATP cleavage and P release are mea sured see below In Table l we summarise examples of different quenching agents that have been used for QF experiments How can intermediates or products be measured The QF technique is a pointbypointmethod To obtain a convincing time course multiple reaction mixtures quenched at different times are needed and each of these has to be analysed In the early days this was hard work but with the advent of automated highperformance liq uid chromatography HPLC equipment coupled with the array of analytical methods now available this problem has to a large extent been resolved As illustrated below the use of y 3ZPATP has facilitated greatly studies on the ATPase and kinase family of enzymes Three further experimental strategies must be consid ered One can work at different enzyme to substrate ra tios ie E lt S multitumover or E gt S single turnover or on different time scales Finally a way to obtain mechanistic information is to experiment over a large temperature range 12 Myosin ATPase is used here as an example to illustrate these strategies Examples A large proportion of QF studies have examined enzymes that use ATP as a substrate This is partlybecause of their biological importance They are also relatively convenient to study since in these systems the time course can often be obtained merely by measuring the concentration of P formed during the reaction or by the quenching process Myosin ATPase is an excellent model to illustrate the ver satility of the QF method Information about the different steps on the reaction pathway of the enzyme was obtained by varying the myosin to ATP ratio to give either mul tiple or singleturnover reactions the time scale of the experiment and the quenching agents These different strategies are summarized in Table 2 Myosin ATPase The QF method was of crucial importance in the initial elucidation of the reaction pathway for myosin ATPase In their pioneering work Lymn and Taylor 13 studied the kinetics of P formation during the hydrolysis of ATP by myosin Reaction mixtures aged from 5 ms upwards were quenched in acid and the total P measured ie enzyme bound as well as free P The time course was biphasic a rapid rise or P burst followed by a steady 2574 T E Barman et al Table 1 Examples of quenching agents other than acid see also ref 7 The quench ow method Agent Observations Examples Chemical Strong alkali used when intermediates of interest are unstable in nucleoside diphosphate lcinase 33 acid phosphoglucomutase SDS protein denaturation without brutal change in pH certain enzymes acting on DNA kinetics of interaction of myosin with EDCactivated actin 6l EDTA stops divalentmetaldependent reactions certain enzymes acting on DNA ATP sulphurylase Excess unlabelled substrate substrate chase Nethylmaleimide in acetic acid Physical Filtration Rapid freezing Rapid evaporation stops binding of radioactive substrate under mild c onditions blocks remaining enzyme cysteines in reaction mixture limited to particulate lowturnover systems reaction mixtures squirted into organic solvent at less than 7140 C and studied by eg EPR spray of reaction mixtures subjected to high voltage field ions analysed by mass spectroscopy 62 substrate y PATPmyosin ATPase mitochondrial ATPase 63 substrate 3Hfarnesyl diphosphatezprotein farnesyl transferase 64 ribonucleoside diphosphate reductase 65 Ca binding to membrane CaztATPase 66 GTP hydrolysis by transducin 67 xanthine oxidase 68 nitric oxide synthase 69 5enolpyruvoylshilcimate3phosphate synthase 70 tryptic hydrolysis of a specific ester substrate 71 Table 2 Myosin ATPase information obtained from different types of experiment using the QF method Type of experiment Reaction mixture Time scale Quenching agent Information obtained range P1 burst multiturnover myosin ATP ms to s acid ATP cleavage kinetics P burst single turnover myosin ATP s lkm acid equilibrium constant of ATP cleavage km Cold ATP chase myosin ATP ms to s unlabelled ATP then ATPbinding kinetics and activesite acid titration ADP displacement myosinADP ATP s lkm acid ADP release kinetics In all the experiments y PATP was the substrate and 32PP1 was determined in the quenched reaction mixtures state phase as illustrated below Figure 2 Lymn and Taylor 1 3 proposed that a ternary myosinADPP1 com plex accumulates in the steady state In support of their proposal Taylor and colleagues 14 subjected myosin ATP reaction mixtures to gel filtration and by a rapid separation procedure confirmed that both ADP and P are associated with the myosin Using QF and 180 ex change methods Bagshaw et a1 15 and Webb and Tren tham l 6 studiedthe cleavage step of the myosinATPase reaction and showed that it is freely reversible ie that the binary enzymesubstrate complex MATP is freely interconvertible with the enzymeproducts complex MADPP Taken together these studies are summarized by the LymnTaylor scheme 1 2 3 M ATP 9 MATP e MADPPi gt M ADP Pi Scheme 1 where step 2 is reversible and step 3 is rate limiting since the MADPP complex accumulates in the steady state ATP binding Further QF studies were directed towards the details of the ATPbinding process is this merely dif fusion controlled as implied in Scheme 1 or does it also involve a protein isomerisation step ie an induced fit mechanism 17 as in Scheme 2 M ATP 9 MATP e MATP Scheme 2 where indicates anATPinduced conformational change of the myosin The kinetics of the binding of ATP to myosin were ex amined by the ATP chase method 18 19 Myosin plus y 3ZPATP reaction mixtures were aged in a QF appara tus and quenched in a large molar excess of unlabelled ATP After incubation for 2 min this mixture was then Cell Mol Life Sci Vol 63 2006 39 l l I Stelt1xstat9 rate 08 a 4 Mm osin 25 M 9213 ATP 2 0 6 Seawateth rate M y H y E E g 04 I E ATPOOr S ac1dll a V 32PPi measured 02 0 100 200 300 Time ms Figure 2 ATP chase o and Pi burst D time courses with myosin under multiturnover conditions at 15 OC Steadystate rates were measured from reactions over longer time scales seconds upwards than those shown here The buffer was 50 mM Trisacetate pH 80 150 mM KCl and 2 mM Mg acetate In the ow diagram the con centrations refer to the reaction mixtures at t 0 MO and ATP0 Shown with permission from ref 20 the Biochemical Society quenched in acid and the concentration of 32Pi was de termined Fig 2 The application of this method led to the following conclusions 19 i ATP binds essentially irreversibly confirming previous work 20 ii the bind ing is a twostep process as proposed previously 21 iii the ATP chase can be used to titrate the kinetically competent sites in myosin The ATP chase experiment shown in Figure 2 reveals a rapid exponential rise in P followed by the steadystate phase of ATP hydrolysis kss The amplitude of the rise shows that the myosin used titrated 08 equivalent active site per mole myosin Thus kcat kSS08 where kcat is ex pressed as mole ATP hydrolysed per second per equiva lent active site and kSS as mole ATP hydrolyzed per sec ond per mole myosin The rate constant of the exponential rise increased hyperbolically with the ATP concentration which is evidence that ATP binds in two steps 1 ATP cleavage In Pi burst experiments reaction mixtures are quenched directly in acid and 32Pi determined A Pi burst experiment carried out under the same conditions as the ATP chase is also illustrated in Figure 2 The time course consists of three phases an initial lag a transient burst and finally a steadystate phase The lag phase is a re ection of the ATP binding process and the burst phase the ATP cleavage step On increasing the ATP concentra tion the lag phase diminishes and the rate of the burst phase increases to a limiting plateau as the kinetics of the ATP cleavage step become rate limiting step 2 in Scheme 1 On decreasing the ATP concentration the rate of the burst phase becomes limited by the ATPbinding kinetics so the lag phase again diminishes Pi burst experiments have also been carried out under sin gleturnover conditions ie with myosin in excess of the Review Article 2575 6 M myosin 2 uM y32PATP U 39 acid 32PPi measured Pi ATP 0 molmol 0 20 40 60 80 100 120 Time s Figure 3 Pi burst experiment with myosin under singleturnover conditions at 4 OC ATPO is the ATP concentration in the reac tion mixture at t 0 The buffer was 50 mM Trisacetate pH 74 100 mM potassium acetate and 5 mM KCl Reprinted with per mission from ref 24 Copyright 1992 American Chemical So ciety ATP 21 23 A typical experiment is illustrated in Fig ure 3 it reveals a very rapid transient burst phase of Pi pro duction too fast to measure on the time scale used here followed by an exponential rise to the complete hydrolysis of the ATP The two parameters obtained amplitude of the transient burst and kinetics of the slower rise are indepen dent of the ATP and myosin concentrations The amplitude corresponds to Kzl K2 where K2 is the equilibrium constant of the cleavage step step 2 in Scheme 1 and the rate of the slow phase corresponds to kcat Release of products From the singleturnover Pi burst experiments it appears that Pi release directs the steady state rate under multiturnover conditions is ADP re leased at the same time from MADPPi or is it released from an MADP complex as in Scheme 3 P 1 MADPPi J MADP a M ADP Scheme 3 Two types of experiments were carried out to answer this question 1 ADP displacement with y 32PATP measurement of ADP release kinetics In this approach which is based on a stopped ow method 24 y 32PATP is added to a solution of myosin and ADP ie to an M ADP complex After ageing the reaction mixtures are quenched in acid and 32PPi is determined The rationale is that the ATP can only bind to myosin after the ADP has been released from the M ADP complex A typical experiment is illustrated in Figure 4 together with controls In the absence of ADP there was a rapid burst phase kinetics too fast to measure on the time scale used here followed by the steadystate phase When ADP and y 32PATP were added together to the myosin there 2576 T E Barman et al 3 I I I I I I Controls 10 M y32PATP a 2 IIM myosin i 10 ILM ADP g 2 E E E acid E 1 339 PPi measured 0 1 I l I I I I I 0 50 100 150 200 250 Time s 3 I I I I I I I ADP displacement 2 IIM myosin g 2 10 M ADP 10 ILM y32PATP E B E a z acid 1 A h 39 b 32PPi measured 0 I I I I I 0 50 100 150 200 250 Time s Figure 4 Time courses for Pi bursts under multiturnover condi tions at 4 OC effect of ADP No ADP 0 or ADP as competitive inhibitor El a ADP in MADP displaced by ATP A b The buffer composition was as in Figure 3 our unpublished data was only a small effect on these parameters Fig 4a However when y 32PATP was added to the myosin with ADP ie to MADP the rate of the burst phase was sig nificantly slowed Fig 4b The kinetics of this phase are a function of the rate of release of the products 25 and they show that the kinetics of ADP release are faster than those of Pi 2 Multiturnover Pl burst under cryoenzymic condi tions A powerful way to obtain information on an en zyme reaction pathway is to work under cryoenzymic conditions This technique involves two perturbants temperature and an antifreeze It may permit the accu mulation of reaction intermediates that cannot be ob served under normal conditions by slowing down the kinetics of their formation by a change in a ratelimit ing step or by shifts in equilibria 12 With myosin ATPase there is a change in the ratelimit ing step as the temperature is decreased Pi release above 0 OC ADP release below 0 0C 26 The effect of this switch on the time course of a Pi burst experiment at 15 0C is illustrated in Figure 5 The progress curve is triphasic a biphasic transient phase followed by a slow steadystate phase The initial fast transient is a mani festation of the kinetics of the formation of MADPPi the second transient the Pi release kinetics and the final steadystate phase the ratelimiting release of ADP Thus a single Pi burst experiment at 15 OC gave kinetic in formation on three steps on the myosin ATPase reaction pathway 27 The quench ow method Equot u N 3 uM myosin 30 MM y 32PATP Lquot u 39 acid PiJMJO molmol 32PPi measured 05 0 10 20 30 40 50 Time min Figure 5 Progress curve for P1 burst with myosin under multiturn over conditions at 15 0C with 40 ethylene glycol as antifreeze The buffer was as in Figure 3 Reprinted with permission from ref 28 Copyright 1999 American Chemical Society Kinases A strategy to study the transient kinetics of the kinases is to quench reaction mixtures in acid or alkali and then to measure directly or indirectly phosphorylated product as Pi For example when creatine and 3phosphoglycerate kinase PGK reactions are quenched in strong acid the phosphorylated products are hydrolyzed to give Pi which can then be measured 28 31 CAMPdependent protein kinase which serves as a model for the protein kinase family has also been studied by QF Reaction mixtures with a peptide substrate were quenched in acid and the concentration of phosphopeptide measured directly 32 Nucleoside diphosphate kinase is an example of an en zyme that catalyzes phosphoryl transfer by a pingpong mechanism involving the phosphorylation of a histidine residue in the enzyme Reaction mixtures enzyme ATP were quenched in 015 N NaOH and the phosphoprotein intermediate measured as phosphohistidine 33 The PGK system is discussed here in more detail because it illustrates another strategy equilibrium perturbation Further this enzyme serves as a model for the applica tion of the QF method to a reversible system It catalyses the reaction ATP 3phosphoglycerate PG 9 ADP 13bisphosphoglycerate bPG Although the forward reaction is unfavourable PGK is usually studied in this direction because of the instability of bPG Two types of experiment were carried out with yeast PGK using y 32PATP In both reaction mixtures were quenched in acid and 132PbPG determined as 32PPi 31 In the first type the time course of bPG formation was obtained it consisted of a transient burst of enzyme bound bPG followed by a steadystate phase until the fi nal equilibrium was reached The kinetics of the transient gave information on the putative hingebending motion of PGK 34 In the second type reaction mixtures at equilibrium were perturbed by the injection of ADP the new reaction mixtures aged for different times before fi nally quenching in acid and determining the bPG concen tration As shown in Figure 6 there was a rapid decrease in bPG This was interpreted by the added ADP react Cell Mol Life Sci Vol 63 2006 06 10 uM PGK 50 MM PG 100 M y32PATP 9 A 100 1M ADP acid 9 N bPGPGK0 molmol 32PPi determined 0 20 40 60 80 100 120 Time ms Figure 6 Perturbation of a PGK reaction at equilibrium by ADP at 4 OC The concentrations given are those immediately after mixing At equilibrium gt 100 ms the total bPG concentration was 57 uM The buffer was 20 mM triethanolamine pH 75 100 mM Kacetate 1 mM free Mg acetate and 30 vv methanol Reprinted with permission from 36 Copyright 2005 American Chemical So ciety ing with a PGKbPG complex in the equilibrium mixture to give rise first to a PGKbPGADP complex then to PGKPGATP and finally to free ATP The use of these strategies led to the following conclusions 35 First a binary PGKbPG complex is an important intermediate on the PGK reaction pathway This implies that ADP is released before bPG Second by perturbing reaction mixtures with ADP the PGK reaction can be studied in the physiologically important direction without having to handle the unstable bPG Finally by the use of a global fitting procedure ref 35 and references therein estimates of the kinetic constants of a sevenstep pathway for PGK were obtained Essentially in this procedure the experimental data obtained at different concentrations of ATP bPG bursts or of ADP ADP perturbation were fitted simultaneously assuming the pathway using Sci entist version 20 MicroMath Research The differen tial equations describing the timedependent change in concentration for all the species were entered and time courses were derived by numerical integration Enzymes acting on DNA The QF method has been applied widely to reactions of proteins on DNA Examples include the replication and proofreading activities of DNA polymerases strand separation by helicases ATP hydrolysis by topoisomer ases transcription by RNA polymerase and its regulation by transcription factors DNA cleavage by nucleases and DNA modification by methyltransferases see refs 36 48 and references therein There are at least two reasons why QF has been used so often on these systems First most reactions on DNA are ratelimited by the dis sociation of the enzyme from the final product More over the polymerases the helicases and some other enzymes 36 37 39 44 46 48 act processively and catalyse many consecutive reactions on a single DNA before dissociating In these situations very little infor Review Article 2577 mation about the reaction pathway can be deduced from the steadystate turnover of the enzyme This informa tion can only be obtained by monitoring the individual steps directly Second with few exceptions reactions on DNA are opti cally silent They seldom produce any change in either UV or visible absorption or uorescence so they can not be observed in a stopped ow spectrophotometer The majority of DNA reactions are therefore carried out by incubating the protein and the DNA for the requisite time and then quenching the sample to stop any further reaction Many enzymes that act on DNA need Mg2 and these can often 40 but not always 37 be stopped by using EDTA to chelate the Mg2 Alternatively reactions on DNA can be quenched with acid or alkali or with a reagent that denatures the protein but not the DNA e g phenol or SDS Finally the DNA substrate is separated from the reaction products usually by electrophoresis and the concentrations of each determined The BvaI restriction endonuclease provides an example of this strategy 47 The orthodox restriction enzymes such as EcoRI or EcoRV are dimers of identical subunits that cleave DNA at sites with the same 5 3 sequence in both strands 40 42 BvaI differs from the orthodox by cleaving DNA at a site with different sequences in each strand and by having two different subunits R1 and R2 each specific for a particular strand R2 539 C C T C A G C 339 339 G G A G TTC G 539 R1 The substrate for these experiments was a 4kb plasmid with one recognition site for BvaI Cutting one strand of this DNA converts it from its native supercoiled SC state to the relaxed opencircle OC form while cut ting both strands at the same site yields its linear LIN form Fig 7a The three forms can be separated from each other by electrophoresis Fig 7b In reactions with BvaI at a lower concentration than the DNA when only a small fraction of the DNA is enzyme bound the initial product liberated from the enzyme is LIN DNA rather than nicked OC DNA 47 The steadystate reactions thus reveal only the rate of dissociation from the product cut in both strands and fail to provide any information about the rates at which either the R1 or the R2 subunits cut their respective strands In contrast by using the QF appara tus to examine singleturnover reactions with BvaI in excess of the DNA the transient formation and decay of the nicked OC DNA was observed as an enzymebound intermediate Fig 7c The data in Figure 7c were fitted to the kinetic equations 1 for the reaction scheme SC 0C LIN to obtain apparent rate constants for cutting first one and then the other strand ka and kb respectively The best fit was ob tained with a value for ka about four times larger than 2573 T E Barman et s1 The quenche ow method SC 0C LIN ka kb b o 30 secs 00 V 7 UN st M vCI 25 nM iisc DNA 0 mM Mgch 39 25 1 EDTA slop mix 2 so A 3 SC 0C and LIN E 15 delcnnincd 2T a 1 05 O t 4 o 10 20 30 Time seconds Figure I with one I39ecognie at BvaI st 37 39 75 A er m o 39 EDTA and the DNA analysed by The time 39 M 0c and UN forms on the right 39 39 39 c the optimsi fit is shown With permission of Elsevier that for kb This differs markedly from the homodimeric restriction enzymes like EcoRV and EcoRI Which cleave 391 t 1 L is not limited by the rate at Which the enzyme binds to its recognition site in the DNA rates 40 42 However BvaI is a heterodimer so e different rates With this enzyme might re ect distinct activities by by order of mixing experiments Forexample on adding Mg to a solution containing the EcoRV nuclease and diately a plasmid substrate DNA cleavage started imme reaction Was carried out repeatedly in the QF to collect But When EcoRV Was added to a solution of plasmid and multiple samples of the time point When the OC formWas Mg DNA cleavage started only after a lag phase 40 39 39 Fi 7 l T 39 bind to lysed to detemiine 39 4 Ahn t 80 had been cleaved in the strand cut by the R1 subunit and 20 in the strand cut by R2 Hence the relatively large rate constant k3 is due mainly to the R1 subunit acting on its strand and the smaller rate constant kb due mainly to R2 on the other stmnd 47 The reaction in Figure 7 Was set up by adding MgClZ to a solution containing both the BvaI enzyme DNA The same results Were obtained When BvaI and MgClZ in one solution Were mixed With DNA in another Hence at these concentrations the rate of DNA cleavage its target in the nla mid secondor der kinetics With respect to the enzyme the rate for bind ing to the recognition site must be limited by the initial bimolecular association ofthe enzyme in free solution to any site on the plasmid the subsequent transfer of the en zyme from its initial random site to its final specific site is too rapid to limit the ovemll rate The transfer occurs mainly by multiple dissociationreassociations Within the same DNA rather than by sliding 49 Similar strategies to those illustrated here With restric tion enzymes have been applied to many other reactions Cell Mol Life Sci Vol 63 2006 on DNA For example both the polymerase and the 3 5 ucts of the po ymerase reaction of length n 1 n 2 bases and under appropriate conditions the exonuclease products n 7 1 ases In addition to monitoring the progress of a reaction on sWer other questions about DNAprotein subjected to nonspecific cleavages throughout its length c series of fragments extending from the label to every possible position along the DNA But if a section of the DNA 39 red by a protein that section is DNA sites can be measured on the millisecond time scale 50 In the example shown Fig 8 the Lac repressor Was first mixed in a QF device With L1 operator DNA 41 After various times this solution Was mixed With a third con taining a high concentration of DNase I The resultant mixture then owed into a solution of EDTA to stop the DNase I The DNase I reaction is thus allowed to proceed foras long as it takes the solutionto ow from the second mixing chamberto the EDTA but this Was sufficient for tion rate constant of the protein forits DNA site Was then evaluated Fig 8 The ability to monitor the position of a protein on DNA is y y RNA polymemse 48 RNA polymerase forms a suc cession of complexes at its promoter sites before initiating RNA synthesis It then leaves the promoter and moves 1 m nu l l l 39 39 l 39 Review Article 25 79 amined by attaching a uorescent label to the 5 end of the DNA 52 orby incorporating a uorescent base such as 2aminopurine into the DNA 43 53 In most ofthese cases the optical signal reports on either the association dissociation of the enzyme from the DNA or a structural a Time mantis ii 63 nM Lac Z repressor l0 pM 39 39Plend labelled DNA DNase I owed into 10 quotIM EDTA lPlDNA fragments analysed chlional Salumiou i i l 1 tzo rune seconds of its reaction 36 By using a rapid QF mixing device coupled to a novel ultrafast footprinting procedure the precise position of the RNA polymerase on t e DNA has b ped at every stage of its reaction 48 Not all reactions on DNA are optically silent Some can be monitored from the hypochromicity of DNA the fact that singlestranded DNA has a higher extinction at 260 u a L1 i nxl 11 Figure 3i QF analysis oflae iepiessor binding to its Operator 42 39 tr QF foo rin in t39p t g ex periment measuring the kineti so Lao p ssorbindmgtn a 18 7 bp DNA fragment witha lac operator ite 0 T time intervals betw l ixingo withthe operator DNA d the subsequent addition ofth ase I are noted a each he section ofthe D mg the operator site is as marked on the right 1 The autoradlogram in a was ana sed to deter e the e e ofprotectio mi o te at each time p t gi e p e shown The solid e is the best fit a th s single exponential Shown with permission ofthe American Socie 2580 T E Barman et al Table 3 Further examples of systems that have been studied by QF The quench ow method Enzyme Reaction Procedure Information obtained Reference Aspartate Laspartate carbamyl phosphate reaction mixtures i regulatory in the absence of regulatory 72 transcarba carbamyl aspartate subunit quenched in acid and subunit no transient phase in mylase carbamyl aspartate measured its presence transient lag phase kinetics of an allosteric transition ATP 1 ATP SOA adenosine 5 enzyme preincubated with AMP reactions 1 and 2 are coupled 62 sulphurylase phosphosulphate APS PP PP and Mg mixed with GTP tightly to drive APS formation 2 T quenched in EDTA and P and fivestep mechanism of GTP GDP determined hydrolysis by EAMPPP com plex Ribonuclease cytidine 2 3 cyclic phosphate quenching problem RNase stable profiles of product progress 73 RNase C gt p 3 CMP hydrolysis C gt p and CpC gt p unstable in curves suggest connection or cyti ylyl3 5 cytid1n acid reaction mixtures quenched between synthesis and hydrolysis cyclic phosphate CpC gt p in pH 2 buffer with pepsin which synthesis stops and then inactivates RNase irreversibly by cleaving Phe120 Asp 12 1 bond CTP ATP UTP glutamine reaction mixtures i GTP quenched without GTP time course had a 75 synthetase ADP CT P glutamate P in acid and P determined P burst with GTP no transient mechanism for CTP formation involves phosphorylation before NH3 attack rather than the reverse as in 74 Phenylala ATP LPhe tRNAPhe enzyme preincubated with time course of TyrtRNAPhe pro 76 ninetRNA AMP PP PhetRNAPhe tyrosine and ATP mixed with duction rapid rise then decay in ligase tRNAPhe quenched in acid and lOms time range as TyrtRNAPhe TyrtRNAPhe measured hydrolyzed shows fidelity of overall process for PhetRNAPhe production rearrangement of the proteinDNA complex rather than the chemical step of the reaction pathway Consequently even when an optical signal is available additional infor mation can be obtained by carrying out parallel experi ments in the QF to observe the chemical step For example the cleavage of a 12bp DNA into 6bp products yields an increase in A260 if the reaction is car ried out at a temperature below the Tm melting point of the substrate but above that for the products the substrate is then doublestranded while the products melt to single strands 51 When EcoRV reactions on a 12bp substrate were examined by this method and by QF the increase in A260 was not coincident with DNA cleavage as measured by QF but occurred after it upon dissociation of the cleaved DNA 54 Presumably the 6bp products remain double stranded while they remain in the DNAbinding cleft of the protein and only melt to single strands in free solution Reactions on the nucleotide bases in DNA are often monitored using 2amjnopurine as a reporter as it has a low uorescence when inside the double helix but a high uorescence when exposed to solvent 43 53 In many ofthese cases the enzyme ips a base out ofthe DNA into its active site where it carries out a reaction on that base In these cases the uorescence from 2aminopu rine reveals the base ipping step in the reaction pathway 53 but quench methods are then needed to observe the subsequent reaction on the exposed base 38 Further QF studies We have selected five examples from the large number of QF studies in the literature that are of particular interest because of the use of a special procedure or the results ob tained These five examples are summarised in Table 3 To conclude multienzyme systems 7 even whole cells 7 can be studied by QF provided that control experiments are carried out and if necessary precautions taken Thus the fragile fatty acid synthetase complex was studied successfully by QF after appropriate measures had been taken 55 Myofibrils are fibrous structures typically 1 mm diameter and 30 mm length that appear not to be damaged when mixed with buffer in a QF apparatus 5 6 and several studies have been carried out on their ATPases ref 57 and references cited therein The sugar uptake of whole blood cells has been studied by QF 58 Conclusions Here our intention was to show the importance of the QF method in the study of enzyme mechanisms With the array of analytical methods currently available and Cell Mol Life Sci Vol 63 2006 by the use of different quenching agents it is a highly versatile method Thus unambiguous information on the chemical and kinetic properties of the intermediates that make up enzyme reaction pathways can be obtained it can not only report on product release as suggested by Fisher 9 but also on essentially all of the preceding steps in the pathway We have illustrated its importance in the study of the mechanisms of ATPhandling enzymes such as myosin ATPase and phosphoglycerate kinase and in determining the 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