Chances Stopped Flow Technique Revolutionized the Investigation of Moderately Fast Reactions

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To eliminate the need for large volumes of reactants and to increase the versatility of measurement, Chance (1943) invented the stopped-flow apparatus by adding a third "stopping" syringe to receive the mixed reactant solutions after passage through the mixer and observation cuvette (see Fig. 10.4). The two reactant syringes are attached to a pneumatic piston that is driven by compressed nitrogen gas. As the third syringe fills, its retreating plunger contacts an electronic switch, stopping the flow of compressed gas and triggering the electronics needed for data acquisition. Importantly, the stopping syringe conserves precious reactants by limiting the reactant volumes needed for each measurement.

FIGURE 10.4 Stopped-flow kinetics apparatus. A, Schematic diagram. A pneumatic driver rapidly forces the plungers forward, driving reactants within syringes A and B through a multi-jet mixer. The mixed solution passes through an observation port (which can be arranged for either absorbance or fluorescence measurements) and into a stopping syringe. The backward motion of the plunger in the stopping triggers data acquisition. B, Applied Physics SX.18MV-R stopped-flow apparatus, providing the basic platform for high performance stopped flow research. In its standard form, the instrument provides a single mixing, single wavelength absorbance and fluorescence detection capability of outstanding sensitivity. Reproduced with permission.

FIGURE 10.4 Stopped-flow kinetics apparatus. A, Schematic diagram. A pneumatic driver rapidly forces the plungers forward, driving reactants within syringes A and B through a multi-jet mixer. The mixed solution passes through an observation port (which can be arranged for either absorbance or fluorescence measurements) and into a stopping syringe. The backward motion of the plunger in the stopping triggers data acquisition. B, Applied Physics SX.18MV-R stopped-flow apparatus, providing the basic platform for high performance stopped flow research. In its standard form, the instrument provides a single mixing, single wavelength absorbance and fluorescence detection capability of outstanding sensitivity. Reproduced with permission.

Ideally, the dead-time (i.e., period required for mixing and stopping flow) is very short, such that the instrument captures the entire reaction time-course. The more realistic outcome is that the 0.7 to 4-msec dead-time overlaps with the reaction kinetics, occasionally obscuring the chemical reaction and frequently mandating some correction. A reaction having a 3- to 4-msec half-life (corresponding to a first-order rate constant of 170 s-1), for instance, would be almost half over if the dead-time were also 3-4 msec. A dead-time of 1-2 msec sets a practical upper limit of 700 sec-1 on the first-order rate constants observable in a stopped-flow experiment.

Another practical consideration is the small reaction volumes (often as low as 0.02-0.1 mL) that is limited only by the required path length in the reaction cell. The BeerLambert absorbance law (see Section 4.4.2) requires that DA = ecl, where DA is the absorbance change at a specified wavelength, e is the molar absorptivity of the absorbing species at the same wavelength, c is the molar concentration of the absorbing species, and l is the path length. Therefore, the signal strength is directly proportional to the path length, and the dimensions of the observation cell strongly affect the experimentally observed signal/noise ratio. Fluorescence detection often allows researchers to improve sensitivity by 100-500 x.

Commercial stopped-flow instruments are "turn-key" devices fitted with modern computer software that controls the machine's basic operating functions (e.g., filling syringes, initiating a reaction, flushing and refilling syringes, etc.) as well as data acquisition, storage and analysis. In practice, the experimenter obtains two types of information: (a) time-dependent changes in signal intensity, corresponding to a record of the reaction's time-evolution; and (b) the signal's amplitude, the latter providing a valuable amount-of-substance information (Table 10.2). To observe the formation and decay of a transient intermediate, the absolute amplitude of change is used to evaluate formation and decay rate constants from information defining the concentrations of reactant(s) or intermediates. For this reason, it is imperative to confirm that the stopped-flow instrument is delivering the proper volumes of each reagent from its two or more syringes, and to be certain that the mixers are scrupulously free of flow-obstructing precipitates/particles. Fortunately, it is relatively easy for the operator to run a test experiment using an acid- or baselabile reagent with well-behaved spectral properties and already determined rate constants. The reagent is placed in syringe A and the acid or base in syringe B, and the rate and amplitude are recorded after mixing. This process is then repeated, except that the reagent is now placed in syringe B and the acid or base in syringe B. An obstructed mixer will result in inefficient mixing, as evidenced by a non-exponential reaction progress curve.

Recognizing the considerable advantages of a stopped-flow apparatus that does not depend on an optical signal output, Howarth, Millar and Gutfreund (1987) invented a stopped-flow calorimeter of sufficiently high sensitivity to monitor heat evolved/consumed in biochemical reactions. Their clever design and suggested operating conditions minimize the effects of work heat (i.e., the heat produced frictionally as viscous solutions pass through delivery tubing and mixing chambers). The thermopile is not fully isolated, and some heat is transferred to the thermopile, to the upstream liquid in the delivery tubing, and to downstream liquid exiting the thermopile. Even so, the conductors within the thermopile are sufficiently efficient to provide accurate thermal kinetic measurements. In a test experiment, they studied the time-course of heat production during a single turnover of chymotrypsin-catalyzed hydrolysis of acetyltyrosine ethyl ester and obtained a first-order rate constant khydrolysis equal to 20 s_1. Millar, Howarth and Gutfreund (1987) subsequently studied the enthalpy changes during individual reaction steps of the ATPase reaction catalyzed by the S-1 myosin subfragment. At 5°C and pH 7.0, the endothermic on-enzyme ATP-cleavage step was observed directly (DH = + 64 kJ-moP1). ADP binding was found to be attended by a biphasic enthalpy change. The release and uptake of protons was investigated by the use of two buffers with widely different heats of ionization, and Howarth, Miller and Gutfreund (1987) found that protons are involved in all four principal steps of the ATPase activity of myosin subfragment S1.

TABLE 10.2 Some Applications of the Stopped-Flow Kinetic Techniques Enzyme Comments

Elastase

(Fink and Meehan, 1979)

H2O2 reaction with hemin

(Kiremer, 1989)

Carbonic anhydrase

Dual-specific protein-tyrosine phosphatases

(Denu and Dixon, 1995)

Xanthine dehydrogenase

(Harris and Massey, 1997)

RecA

(Bazemore, Takahashi and Radding, 1997)

Nitric oxide synthase

(Abu-Soud etal, 1999)

Group-I intron ribozyme

(Silverman and Cech, 2001)

Tetrahedral intermediates were detected using specific chromogenic di- and tripeptide p-nitroanilide substrates as they accumulated at high pH, using sub-zero temperatures and aqueous/organic cryosolvents. The tetrahedral adducts are characterized by spectra with 1max of 359 ± 2 nm, compared with that of 380 nm for the product p-nitroaniline and 315-320 nm for the substrates. The pH dependence for formation of the tetrahedral intermediates exhibited rate-limiting conditions at low pH, and collapse to the acyl-enzymes was rate-determining at higher pH.

The kinetics of formation of the dominant intermediate formed between hemin and H2O2 was investigated by the stopped-flow method. CII formation is preceded bya precursor CI, for which a steady-state concentration was established early in the reaction. Relevant rate constants (or combinations of them) and the molar absorption coefficients of the intermediates at 400 nm were determined.

Human carbonic anhydrase II (HCA II) catalysis of CO2 hydration includes the transfer of a proton from zinc-bound water to histidine 64 via a network of intervening hydrogen-bonded water molecules, whereupon the proton is transferred to solution-phase buffer. The authors used stopped-flow spectrophotometry and 18O exchange between CO2 and water measured by mass spectrometry to compare catalytic constants dependent on proton transfer in HCA II and in the mutant His-64-Ala HCA II containing the replacement His64Ala. The following parameters were very similar or identical in catalysis by His-64-Ala HCA II compared with catalysis by wild-type HCA II both in the presence of large concentrations of imidazole (100 mM): the maximal rate of initial velocity and of exchange of 18O between CO2 and water, solvent hydrogen isotope effects on the maximal velocity, and the dependence of these isotope effects on the atom fraction of deuterium in solvent water. These results indicate that the proton transfer involving the zinc-bound water in catalysis is not significantly affected by the difference between the mobility of the free imidazole buffer and the side chain of His-64.

In view of the fact that these enzymes have a common active-site sequence HCXXGXXRS(T), the authors examined the role of the conserved hydroxyl by changing Ser-131 to Ala. The pH profile of kcat/Km for the Ser-131-Ala mutant is indistinguishable from that ofthe native enzyme, but the kcat value for Ser-131-Ala mutant is 100-fold lower than that for the native enzyme. The shape ofthe pH profile was perturbed from bell-shaped in the native enzyme to a pH-independent curve over the 4.5-9.0 pH range, suggesting the mutation alters the rate-limiting step. Loss of this hydroxyl group at the active site dramatically diminished the ability the thiol-phosphate intermediate to hydrolyze without exerting any significant change in the steps leading to and including intermediate formation.

The reaction between pre-reduced XDH and NAD+, as studied by stopped-flow spectrophotometry, was found to involve two rounds of oxidation with 2 eq of NAD+. The first round goes to completion, and the second round reaches a slightly disfavored equilibrium. Rapid binding of NAD+ with an apparent Kd of 25 ± 2 mM is followed by NAD+ reduction at a rate constant of 130 ± 13 s-1. NADH dissociation at a rate constant of 42 ± 12 s-1 completes a round of oxidation.

The authors measured RecA-catalyzed pairing and strand exchange in solution by energy transfer between fluorescent dyes on the ends of deoxyribo oligonucleotides. By varying the position of the dyes in separate assays, the authors detected the pairing of single-stranded RecA filament with duplex DNAas an increase in Forster resonance energy transfer, and strand displacement as a decrease in FRET.

The binding kinetics of L-arginine and its alternative substrates homoarginine, N-methylarginine, and N-hydroxyarginine to nNOS were characterized by conventional and stopped-flow spectroscopy. Because binding of these substrates results in only a small light absorbance change in tetrahydrobiopterin-saturated nNOS, binding was monitored by following displacement of imidazole, which displays a significant change in Soret absorbance from 427 to 398 nm. Rates of spectral change upon mixing Imidazole-nNOS complex with increasing amounts of substrates were obtained and found to be mono-phasic in all cases.

Folding of the 160-nt P4-P6 domain of the Tetrahymena group-I intron RNA involves burying of substantial surface area. Stopped flow fluorescence was used to monitor the Mg2+-induced tertiary folding of pyrene-labeled P4-P6. At 35°C with [Mg2^ ~10 mM, P4-P6 folds on the tens of millisec time-scale with kobs = 15-31 s-1. The kobs depends on [Mg2+], and the initial slope of kobs versus [Mg2+] suggests that only approximately one Mg2+ ion is bound in the rate-limiting folding step. This is consistent with an early folding transition state, because folded P4-P6 binds many Mg2+ ions. The observation of a substantial DGz despite an early folding transition state suggests that a simple two-state folding diagram for Mg2+-induced P4-P6 folding is incomplete. These studies provided quantitative estimates for the activation barrier and location of a transition state for tertiary folding of an RNA domain.

Mudd and Berger (1988) also described a differential stopped-flow, heat conduction calorimeter with mJoule resolution. This all-tantalum instrument consists of two matched channels, each having two reagent inlet lines, a thermopile, and a computer that processes the data and controls the syringe-drives (mixing dead-time = 0.6 sec). Although a poorer heat conductor than copper (e.g., 0.5 Watts/cm$K for the former and 4 Watts/cm$K for the latter), tantalum is highly resistant to chemical corrosion.

Porschke (1998) recently described a stopped-flow field-jump instrument for analyzing changes in macro-molecular structure during the course of various biochemical reactions. The device takes advantage of a new type of cell construction where the electrodes are integrated within the quartz cuvette. The instrument is very conservative in terms of sample quantities and offers a high time-resolution. The dead-time for mixing is approximately 0.5 msec, and electric field pulses with field strengths up to 20 kV/cm can be applied with rise-times in the nanosecond range. The time resolution of the optical detection is up to the nanosecond time range. To illustrate the utility of this approach, Porschke (1998) examined the intercalation of ethidium bromide into double-helical DNA. At a wavelength of 313 nm, the observed transients are exclusively attributable to DNA-bound ethidium, and there is a relatively high negative dichroism observable at 0.5 msec after mixing. The absolute value of this negative dichroism increases in the millisecond time range, approaching equilibrium within one second. Observed decay time constants for this dichroism demonstrate an increased effective DNA length upon ethidium binding, already evident within 0.5 msec after mixing. A further increase in length occurs over the millisecond time range where equilibrium value is attained. Depending on experimental conditions, there appear to be as many as three discernible relaxation processes.

10.2.3. NAD+ Binding to Alcohol Dehydrogenase: A Case Study Illustrating the Utility of the Stopped-Flow Kinetic Measurements

Recognizing the pedagogical value of an excellent example, let us consider the case of alcohol dehydrogenase (EC 1.1.1.1), which catalyzes the reversible hydride transfer from an alcohol to NAD to produce an aldehyde (or ketone) along with NADH and a proton. Substrates for the horse liver enzyme include a wide variety of primary or secondary alcohols, including ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, cyclohexanol, and even benzyl alcohol. For some time, the ease of ADH activity assays plus a wealth of early structural and kinetic data from Nobel Laureate Hugo Theorell's laboratory - supplemented by the later X-ray crystal structure from Carl Branden's laboratory - has made the horse liver enzyme a model system for experimental testing of the underlying theory for initial-rate and fast kinetics, abortive complex formation, equilibrium isotope exchange kinetics, as well as kinetic isotope effects and tunneling.

Another fascinating aspect of alcohol dehydrogenase is that each subunit contains two zinc ions, one located within the active site, and the second bound to four cysteinyl thiol groups in a manner stabilizing a loop structure that maintains the enzyme's quaternary structure. Although earlier studies had attributed the pKa values of 9.2 and 7.6 to ionization of the zinc-bound water in the active site, microscopic assignment to a particular functional group is hampered by the fact that the zinc-bound water is only one part of a manifold of hydrogen-bonded groups serving as proton relay systems connecting the alcohol substrate and His-51 within the active site. In the study described at length here, Kovaleva and Plapp (2005) demonstrated that NAD binding to the enzyme is limited by a unimolecular step that is likely to be linked to a conformational change of the Enz • NAD+ complex.

These investigators analyzed NAD binding kinetics with a stopped-flow instrument (dead-time = 2.4 ms). To prevent catalysis, coenzyme binding was carried out in the presence of three inhibitors known to coordinate to the active-site zinc ion: (a) pyrazole, the five-member heterocycle known also as 1,2-diazole; (b) caprate (also known as n-decanoate); and (c) 2,2,2-trifluoroethanol, an alcohol that is incapable of hydride transfer. NAD binding in the presence of excess pyrazole was detected by the 293-nm absorbance increase that attends formation of the Enz NAD + Pyrazole complex. The rate of coen-zyme binding was also examined by quantifying the quenching of the enzyme's intrinsic fluorescence (excitation at 293 nm, and emission measured over 310-384 nm). Transient fluorescence data were corrected for the inner-filter effect by NAD . Coenzyme binding in the presence of caprate was also examined by an increase in an absorbance change at 280 nm, as observed in the difference spectrum of the Enz NAD + Caprate complex. Because ADH also absorbs strongly at 280 nm (A = 0.455 mg"J-mL-cm), NAD + concentrations were restricted to below 0.22 mM to keep the total absorbance below 1.2. Proton release (or uptake) was followed by the decrease (or increase) in absorbance of phenol red (at 559 nm) and thymol blue (at 596 nm) using weakly buffered solutions. Enzyme solutions used for stopped-flow experiments were also titrated to determine the number of protons released per active site of ADH during formation of the Enz • NAD + • Inhibitor complex.

A major conclusion of their study was that the deproto-nation of the Enz NAD + complex is kinetically coupled to the rate-limiting step preceding the formation of the ternary complexes. A general mechanism and rate constants for the

Kovaleva and Plapp (2005), this mechanism resembles previously published mechanisms, except that their new results evaluate the rate constants for the steps leading to the ternary complexes (Table 10.3), thereby defining the major pathway (shown in blue in Scheme 10.1) for ternary complex formation.

Based on these rate studies, deprotonation of the Enz NAD+ complex (pKa = 7.3) is characterized by a rate constant of about 200-550 s-1 and represents a common, rate-limiting unimolecular step in the mechanisms of coenzyme binding in the presence of three different inhibitors. The biphasic proton transfer kinetics with the anionic inhibitor caprate was particularly informative, because proton release was followed by proton uptake, and the kinetic steps can be distinguished. In contrast, one proton was lost during formation of the ternary complexes with pyrazole and trifluoroethanol, and the continuous release of protons was best analyzed with simulations of the entire reaction progress curves. The authors suggest that proton is probably released from the water bound to the catalytic zinc in the Enz-NAD+ complex, producing hydroxide that is displaced later during ternary complex

TABLE 10.3 Summary of Estimated Rate Constants for Transient Reactions of Alcohol Dehydrogenase with NAD+ and Selected Inhibitors3

Parameter

Caprated

Pyrazole

2,2,2-Trifluoroethanol

K1 (mM)b

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