Chemical Quench-flow Methods Research Articles

Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein

Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic information including active site concentrations and intrinsic rate constants that govern macromolecular function. In case of enzymes, for example, transient or pre-steady state measurements identify and characterize individual events in the reaction pathway, whereas steady state measurements only yield overall catalytic efficiency and specificity. Individual events such as protein-protein or protein-ligand interactions and rate-limiting conformational changes often occur in the millisecond timescale, and can be measured directly by stopped-flow and chemical-quench flow methods. Given an optical signal such as fluorescence, stopped-flow serves as a powerful and accessible tool for monitoring reaction progress from substrate binding to product release and catalytic turnover(1,2). Here, we report application of stopped-flow kinetics to probe the mechanism of action of Msh2-Msh6, a eukaryotic DNA repair protein that recognizes base-pair mismatches and insertion/deletion loops in DNA and signals mismatch repair (MMR)(3-5). In doing so, Msh2-Msh6 increases the accuracy of DNA replication by three orders of magnitude (error frequency decreases from approximately 10(-6) to 10(-9) bases), and thus helps preserve genomic integrity. Not surprisingly, defective human Msh2-Msh6 function is associated with hereditary non-polyposis colon cancer and other sporadic cancers(6-8). In order to understand the mechanism of action of this critical DNA metabolic protein, we are probing the dynamics of Msh2-Msh6 interaction with mismatched DNA as well as the ATPase activity that fuels its actions in MMR. DNA binding is measured by rapidly mixing Msh2-Msh6 with DNA containing a 2-aminopurine (2-Ap) fluorophore adjacent to a G:T mismatch and monitoring the resulting increase in 2-aminopurine fluorescence in real time. DNA dissociation is measured by mixing pre-formed Msh2-Msh6 G:T(2-Ap) mismatch complex with unlabeled trap DNA and monitoring decrease in fluorescence over time(9). Pre-steady state ATPase kinetics are measured by the change in fluorescence of 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin)-labeled Phosphate Binding Protein (MDCC-PBP) on binding phosphate (Pi) released by Msh2-Msh6 following ATP hydrolysis(9,10). The data reveal rapid binding of Msh2-Msh6 to a G:T mismatch and formation of a long-lived Msh2-Msh6 G:T complex, which in turn results in suppression of ATP hydrolysis and stabilization of the protein in an ATP-bound form. The reaction kinetics provide clear support for the hypothesis that ATP-bound Msh2-Msh6 signals DNA repair on binding a mismatched base pair in the double helix. F

Pre-Steady-State Kinetic Analysis of the Elongation Mode of Dengue Virus RNA Polymerase Domain

Dengue viral RNA polymerase replicates its positive single-stranded RNA genome in a primer-independent manner. The slow and inefficient initiation during replication masks the elongation mode. The aim of this work was to further characterize the mechanism of elongation towards an increased understanding of how the enzyme selectively recognizes different nucleotides. Transient kinetic methods were used to measure the microscopic rates of the reaction pathway comprised of enzyme and RNA binding followed by nucleotide binding and incorporation. After extended pre-incubation of the enzyme with double stranded RNA (12-mer primer with a 26-mer template), addition of a correct nucleotide resulted in a burst of single nucleotide incorporation, and the amplitude of the reaction was used to monitor the time course of RNA binding. The kinetics of enzyme and RNA binding followed a two-step mechanism: an initial binding was followed by a conformational change. The enzyme and RNA binding proceeded to equilibrium about six times faster at 37 oC than at 30 oC, suggesting the conformational change involved in RNA binding is temperature sensitive. Following incubation of RNA and enzyme to form an active complex, we used chemical quench-flow methods to measure the ground state binding Kd and the incorporation rate of a correct nucleotide. The fidelity of the polymerase was determined by measurement of the incorporation of incorrect nucleotides. Using these methods, we characterized the Dengue polymerase in its elongation mode by measuring the kinetics of RNA and polymerase binding and the kinetics for nucleotide binding and incorporation. Based on these data, we built a working model for studying the selectivity of Dengue polymerase.

Investigation of enzyme kinetics using quench-flow techniques with MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is combined off-line with rapid chemical quench-flow methods to investigate the pre-steady-state kinetics of a protein-tyrosine phosphatase (PTPase). PTPase kinetics are generally interrogated spectrophotometrically by the employment of an artificial, chromophoric substrate. However, that methodology places a constraint on the experiment, hampering studies of natural, biochemically relevant substrates that do not incorporate a chromophore. The mass spectrometric assay reported herein is based on the formation of a covalent phosphoenzyme intermediate during substrate turnover. This species is generated in the reaction regardless of the substrate studied and has a molecular weight 80 Da greater than that of the native enzyme. By following the appearance of this intermediate in a time-resolved manner, we can successfully measure pre-steady-state kinetics, regardless of the incorporation of a chromophore. The strengths of the mass-spectrometric assay are its uniform response to all substrates, simple and direct detection of covalent enzyme-substrate intermediates, and facile identification of enzyme heterogeneities that may affect enzymatic activity.

Kinetic Mechanism of GTP Binding and RNA Synthesis during Transcription Initiation by Bacteriophage T7 RNA Polymerase

We have used stopped-flow and rapid chemical quench-flow methods to investigate the kinetics of the early steps during transcription initiation by bacteriophage T7 RNA polymerase. Most promoters of T7 RNA polymerase initiate with two GTPs. The kinetics of GTP binding was investigated by monitoring the fluorescence changes resulting from GTP binding to polymerase and fluorescent 2-aminopurine-containing promoter DNA complex. Scheme 1 was determined from studies of T7 Phi10 promoter at 25 degrees C, where (E.D)n represents the polymerase.DNA complex in different conformations. GTPE and GTPI represent the elongating and initiating GTP molecules incorporated at the +2 and +1 positions, respectively. Our studies show that GTP at the elongation site binds with at least 10-fold tighter affinity than the GTP at the initiation site. Two conformational changes were revealed upon GTP binding to the polymerase.2-aminopurine DNA complex. The first conformational change occurred upon GTP binding to the elongation site. This conformational change was reversible, and studies with partially melted DNA and incorrect NTPs suggested that it may represent a DNA melting and/or base pairing step. A second rate-limiting conformational change whose rate was same as the maximum rate of pppGpG synthesis occurred after two GTPs were bound. As with DNA polymerases, this rate-limiting conformational change probably occurs at each NMP incorporation event and may be involved in proper positioning of the initiation and the elongating GTPs within the polymerase active site to achieve efficient and accurate RNA synthesis.

Catalytic mechanism of KDO8P synthase. Pre-steady-state kinetic analysis using rapid chemical quench flow methods

The catalytic mechanism of 3-deoxy- d -manno-2-octulosonate-8-phosphate (Kdo8P) synthase from Escherichia coli was investigated under pre-steady-state conditions using rapid chemical quench flow methods. The results suggest the formation of acyclic bisphosphate 1 as a reaction intermediate. The catalytic mechanism of 3-deoxy- d -manno-2-octulosonate-8-phosphate (Kdo8P) synthase from Escherichia coli was investigated under pre-steady-state conditions using rapid chemical quench flow methods. The results suggest the formation of acyclic bisphosphate 1 as a reaction intermediate.

Mutants affecting nucleotide recognition by T7 DNA polymerase.

Analysis of two mutations affecting nucleotide selection by the DNA polymerase from bacteriophage T7 is reported here. Two conserved residues (Glu480 and Tyr530) in the polymerase active site of an exonuclease deficient (exo-) T7 DNA polymerase were mutated using site-directed mutagenesis (Glu480-Asp and Tyr530-Phe). The kinetic and equilibrium constants governing DNA binding, nucleotide incorporation, and pyrophosphorolysis were measured with the mutants E480D(exo-) and Y530F(exo-) in single-turnover experiments using rapid chemical quench-flow methods. Both mutants have slightly lower Kd values for DNA binding compared to that of wild-type(exo-). With Y530F(exo-) the ground state nucleotide binding affinity was unchanged from wild-type for dGTP and dCTP, was 2-fold lower for dATP and 8-10-fold lower for dTTP binding. With E480D(exo-), the binding constants were 5-6-fold lower for dATP, dGTP, and dCTP and 40-fold lower for dTTP binding compared to those constants for wild-type(exo-). The significance of a specific destabilization of dTTP binding by these amino acids was examined using a dGTP analog, deoxyinosine triphosphate, which mimics the placement and number of hydrogen bonds of an A:T base pair. The Kd for dCTP opposite inosine was unchanged with wild-type(exo-) (197 microM) but higher with Y530F(exo-) (454 microM) and with E480D(exo-) (1 mM). The Kd for dITP was the same with wild-type(exo-) (180 microM) and Y530F(exo-) (229 microM), but significantly higher with E480D(exo-) (3.2 mM). These data support the suggestion that E480 selectively stabilizes dTTP in the wild-type enzyme, perhaps by hydrogen bonding to the unbonded carbonyl. Data on the incorporation of dideoxynucleotide analogs were consistent with the observation of a selective stabilization of dTTP by both residues. Pyrophosphorolysis experiments revealed that neither mutation had a significant effect on the chemistry of polymerization. The fidelity of the mutants were examined in misincorporation assays. Both E480D(exo-) and Y530F(exo-) showed saturation kinetics with the wrong nucleotide, with binding constants of 1-3 mM compared to the estimated binding affinity of 6-8 mM with wild-type(exo-). Accordingly, both mutants showed slightly lower selectivity against misincorporation. Taken together, these results indicate that E480 and Y530 each contribute to ground state nucleotide binding and suggest that the E480 may serve to specifically stabilize the incoming dTTP of A:T base pairs to compensate for the fewer hydrogen bonds compared to G:C base pairs.

Pre-steady-state kinetics of the microtubule-kinesin ATPase.

The pre-steady-state kinetics of the microtubule-kinesin ATPase were investigated by chemical-quench flow methods using the Drosophila kinesin motor domain (K401) expressed in Escherichia coli [Gilbert, S. P., & Johnson, K. A. (1993) Biochemistry 32, 4677-4684]. The results define a minimal mechanism: M.K + ATP in equilibrium with (M).K.ATP in equilibrium with (M).K.ADP.Pi in equilibrium with M.K.ADP + Pi in equilibrium with M.K + ADP, where M, K, and Pi represent microtubules, kinesin, and inorganic phosphate, respectively, with k+1 = 0.8-3 microM-1 s-1, k-1 = 100-300 s-1, k+2 = 70-120 s-1, k+4 = 10-20 s-1, and k+3 >> k-2 and k+3 >> k+4. Conditions were as follows: 25 degrees C, 20 mM HEPES, pH 7.2 with KOH, 5 mM magnesium acetate, 0.1 mM EDTA, 0.1 mM EGTA, 50 mM potassium acetate, 1 mM DTT. The experiments presented do not determine the step in the cycle where kinesin dissociates from the microtubule or the step at which kinesin reassociates with the microtubule; therefore, the steps that may represent kinesin as the free enzyme are indicated by (M). A burst of ADP product formation was observed during the first turnover of the enzyme in the acid-quench experiments that define the ATP hydrolysis transient. The observation of the burst demonstrates that product release is rate limiting even in the presence of saturating microtubule concentrations. The pulse-chase experiments define the time course of ATP binding to the microtubule-K401 complex. At low ATP concentrations, ATP binding limits the rate of the burst. However, at high concentrations of ATP, ATP binding is faster than the rate of ATP hydrolysis with k+2 = 70-120 s-1. The amplitude of the burst of the ATP binding transient reached a maximum of 0.7 per site at saturating concentrations of ATP and microtubules. The amplitude of less than 1 is attributed to the fast k(off) for ATP (k-1 = 100-300 s-1) that leads to a partitioning of the M.K.ATP complex between ATP hydrolysis (k+2) and ATP release (k-1). These results indicate that ATP binds weakly to the M.K complex (Kd,ATP app approximately 100 microM). ADP release (k+4 = 10-20 s-1) is rate limiting during steady-state turnover, indicating that microtubules activate the kinesin ATPase by increasing k(off),ADP from 0.01 s-1 in the absence of microtubules to 10-20 s-1 at saturating microtubule concentrations.

61] Rapid kinetic analysis of mechanochemical adenosinetriphosphatases

The analysis of a reaction pathway by stopped-flow and chemical quench-flow methods provides the only means of directly measuring the rates of reaction at the active site of an enzyme. The interpretation of data from each technique is similar and involves solution of the differential equations describing the approach of the intermediates to steady state. In the case of the stopped-flow data, I have tried to show how intuition can simplify some of the math to allow the solution of more complex models and to reveal the form of the concentration dependence of the rates. In the case of the chemical quench-flow data, more effort is expended in collecting one time course and accordingly more time is spent in fitting the data directly to a given model. One often is restricted by the quantities of enzyme available so that it is feasible to perform chemical quench-flow experiments at only one or two carefully selected concentrations of substrate. Stopped-flow and chemical quench-flow experiments complement one another because each provides information that is inaccessible, or at least difficult to obtain, by the other method. Our current chemical quench-flow apparatus, requiring only 20 microliter of sample per time point, should allow many more enzymes to be examined by this method.

The pathway of ATP hydrolysis by dynein. Kinetics of a presteady state phosphate burst.

The kinetics of ATP binding and hydrolysis (formation of acid-labile phosphate) by the Tetrahymena 30 S dynein ATPase has been measured by chemical quench flow methods. The amplitude of the ATP-binding transient gave a molecular weight per ATP-binding site of approximately 750,000, suggesting nearly 3 ATP binding sites/2 million Mr dynein molecule (Johnson, K. A., and Wall, J.S. (1983) J. Cell Biol. 96, 669-678). ATP binding occurred at the rate predicted from the apparent second order rate constant of 4.7 X 10(6) M-1 S-1 measured by analysis of the ATP-induced dissociation of the microtubule-dynein complex (Porter, M. E., and Johnson, K. A. (1983) J. Biol. Chem. 258, 6582-6587). Hydrolysis was slower than binding and occurred at a rate of 55 S-1, at 30 and 50 microM ATP. The rate limiting step for steady state turnover (product release) occurred with a rate constant of 8 S-1. These data show that the first two steps of the pathway of coupling ATP hydrolysis to the microtubule-dynein cross-bridge cycle are the same as those described by Lymn and Taylor for actomyosin (Lymn, R. W., and Taylor, E. W. (1971) Biochemistry 10, 4617-4624). Namely, ATP binding induces the very rapid dissociation of dynein from the microtubule and ATP hydrolysis occurs more slowly following dissociation. Moreover, in spite of rather gross structural differences, the kinetic constants for dynein and myosin are quite similar.

Intermediate states of subfragment 1 and actosubfragment 1 ATPase: reevaluation of the mechanism.

The kinetics of the increase in protein fluorescence following the addition of ATP to subfragment-1 (SF-1) and acto-SF-1 have been reinvestigated. The concentration dependence of the rate obtained with SF-1 did not fit a hyperbola and at high ATP concentration, approximately 40% of the signal amplitude was lost due to a fast phase at the beginning of the transient (20 degrees C). At lower temperature (less than or equal to 10 degrees C) the fluorescence transient was biphasic, with a fast phase observed at high ATP concentration. These results indicate that there are two steps in the SF-1 pathway in which there is a change in protein fluorescence. Measurements of ATP binding and hydrolysis by chemical quench-flow methods indicate that the rate of ATP binding is correlated with the fast fluorescence step and hydrolysis is correlated with the slow fluorescence change. The SF-1 mechanism can thus be described as: (formula: see text) where M represents SF-1 and states of enhanced fluorescence are given by M (16%) and M (36% enhancement, relative to SF-1). Step 1 is a rapid equilibrium with K1 approximately 10(3) M-1. Tight binding of ATP occurs in step 2 and the loss of signal amplitude requires k2 greater than or approximately 1500--2000 s-1. The maximum observed fluorescence rate defines the rate of hydrolysis, k3 + k-3 = 125 s-1 (20 degrees C, 0.1 M KCl, pH 7.0). The steps in the mechanism correspond to the Bagshaw--Trentham scheme, with the important difference that the assignment of rate constant is altered. Formation of the acto-SF-1 complex gave a fluorescence enhancement of approximately 14% relative to SF-1. Dissociation of acto-SF-1 by ATP produced a 20--22% enhancement in fluorescence. There was no detectable fluorescence change during dissociation as evidenced by a lag in the fluorescence transient which corresponded to the kinetics of dissociation. The fluorescence change occurred at the same maximum rate as for SF-1 but there was no loss in signal amplitude at high ATP concentration. The kinetics of the fluorescence change corresponded to the rate of ATP hydrolysis, whereas tight ATP binding occurred at a much faster rate in approximate agreement with the rate of dissociation. Thus the fluorescence change in the acto-SF-1 pathway corresponds to step 3 in the SF-1 mechanism. The complete scheme can be described as follows: (formula: see text) where AM represents acto-SF-1. The tight binding step in the SF-1 pathway (k2) is sufficiently fast so that a similar step (k2') in the acto-SF-1 pathway could precede dissociation but the AM-ATP intermediate has not been detected. Following hydrolysis on the free SF-1, actin recombines with M.ADP.Pi or possibly with a second SF-1 product intermediate as proposed by Chock et al. (1976) and the fluorescence returns to the original AM level with product release.