Kinetics: A Tool to Study Molecular Motors

Gilbert, Susan P.; Mackey, Andrew T.

doi:10.1006/meth.2000.1086

Cited by 69 publications

(85 citation statements)

References 66 publications

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“…Steady-state velocity data was collected as described previously (37). Briefly, reactions containing 50 mM Tris (pH 8.1), 10 mM Mg(OAc) 2 The steady-state velocities were then determined from the linear phase of a plot of the amount of ADP generated versus time using KaleidaGraph (Synergy, Inc.).…”

Section: Radiolabeled Atp Hydrolysis Assaymentioning

confidence: 99%

“…We determined the steady-state kinetic constants associated with ATP hydrolysis by recombinant S. Typhimurium and human Lon using a radiolabeled ATP hydrolysis assay (37). The k obs for steady-state ATP hydrolysis at varying concentrations of ATP both in the presence and absence of saturating 1 or 2 were measured and plotted as a function of ATP (Figure 4).…”

Section: Steady-state Kinetic Analysis Of Atp Hydrolysismentioning

confidence: 99%

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Identification of the Proteasome Inhibitor MG262 as a Potent ATP-Dependent Inhibitor of the Salmonella enterica serovar Typhimurium Lon Protease

2006

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Lon is a homo-oligomeric ATP-dependent serine protease which functions in the degradation of damaged and certain regulatory proteins. The importance of Lon activity in bacterial pathogenicity has led to its emergence as a target in the development of novel antibiotics. As no potent inhibitors of Lon activity have been reported to date, we sought to identify an inhibitor which could serve as a lead compound in the development of a potent Lon-specific inhibitor. To determine whether a nucleotide-or peptide-based inhibitor would be more effective, we evaluated the steady-state kinetic parameters associated with both ATP and peptide hydrolysis by human and Salmonella enterica serovar Typhimurium Lon. Although the ATP hydrolysis activities of both homologs are kinetically indistinguishable, they display marked differences in peptide substrate specificity. This suggests that a peptide-based inhibitor could be developed which would target bacterial Lon, thereby decreasing side-effects due to cross-reactivity with human Lon. Using Salmonella enterica serovar Typhimurium Lon as a model, we evaluated the IC 50 values of a series of commercially available peptide-based inhibitors. Those inhibitors which behave as transition state analogs were the most useful in inhibiting Lon activity. The peptidyl boronate, MG262, was the most potent inhibitor tested (IC 50 = 122 ± 9 nM) and required binding, but not hydrolysis of ATP to initiate inhibition. We hope to use MG262 as a lead compound in the development of future Lon specific inhibitors.The number of pathogenic, antibiotic resistant bacteria increases each year, however the development of new antibiotics to treat them lags behind (1). Recent studies aimed at identifying proteins necessary for virulence have implicated the importance of Lon protease (2,3). Pathogenic Salmonella enterica are responsible for causing a range of human diseases from mild gastroenteritis (serovar Typhimurium and serovar Enteritidis) to typhoid fever (serovar Typhi). It has been demonstrated that Salmonella enteria serovar Typhimurium (S. Typhimurium) Lon protease activity is required for systemic infection in mice, a common study model for S. Typhi infection in humans (3). In fact, Lon-deficient S. Typhimurium, when administered as an oral vaccine to mice, conferred subsequent protection against infection by virulent S. Typhimurium (4). Taken together, these studies highlight Lon as an important target in the development of novel therapeutic agents.Lon, also known as the protease La, is a homo-oligomeric ATP-dependent serine protease, which functions in the degradation of damaged and certain short-lived regulatory proteins (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Homologs exist ubiquitously in nature, however they localize to the cytosol in prokaryotes and to the mitochondrial matrix in eukaryotes (8,15,16 (11,(18)(19)(20)(21)(22)(23).Lon protease is a member of the AAA + superfamily (ATPases Associated with different cellular Activities) along with other ATP-dependent proteases such as ClpXP, H...

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Section: Radiolabeled Atp Hydrolysis Assaymentioning

confidence: 99%

Section: Steady-state Kinetic Analysis Of Atp Hydrolysismentioning

confidence: 99%

Identification of the Proteasome Inhibitor MG262 as a Potent ATP-Dependent Inhibitor of the Salmonella enterica serovar Typhimurium Lon Protease

2006

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“…ApoEg5 steady-state ATPase activity (in the absence of microtubules) was determined by measuring [α-32 P]ADP·P i product formation as described previously (26). In Figure 2, the rate of ATP turnover was plotted as a function of MgATP concentration, and the data were fit to the following quadratic equation: (1) where rate is the concentration of product formed per second per Eg5 site, k cat is the maximum rate constant of product formation at saturating substrate, E 0 is the total Eg5 site concentration, and K m,ATP is the MgATP concentration needed to provide one-half the maximal velocity.…”

Section: Steady-state Atpase Kineticsmentioning

confidence: 99%

ATPase Mechanism of Eg5 in the Absence of Microtubules: Insight into Microtubule Activation and Allosteric Inhibition by Monastrol

Cochran

Gilbert

2005

Biochemistry

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The ATPase mechanism of kinesin superfamily members in the absence of microtubules remains largely uncharacterized. We have adopted a strategy to purify monomeric human Eg5 (HsKSP/ Kinesin-5) in the nucleotide-free state (apoEg5) in order to perform a detailed transient state kinetic analysis. We have used steady-state and presteady-state kinetics to define the minimal ATPase mechanism for apoEg5 in the absence and presence of the Eg5-specific inhibitor, monastrol. ATP and ADP binding both occur via a two-step process with the isomerization of the collision complex limiting each forward reaction. ATP hydrolysis and phosphate product release are rapid steps in the mechanism, and the observed rate of these steps is limited by the relatively slow isomerization of the Eg5-ATP collision complex. A conformational change coupled to ADP release is the rate-limiting step in the pathway. We propose that the microtubule amplifies and accelerates the structural transitions needed to form the ATP hydrolysis competent state and for rapid ADP release, thus stimulating ATP turnover and increasing enzymatic efficiency. Monastrol appears to bind weakly to the Eg5-ATP collision complex, but after tight ATP binding, the affinity for monastrol increases, thus inhibiting the conformational change required for ADP product release. Taken together, we hypothesize that loop L5 of Eg5 undergoes an "open" to "closed" structural transition that correlates with the rearrangements of the switch-1 and switch-2 regions at the active site during the ATPase cycle.Motor proteins from the myosin, kinesin, and dynein superfamilies are important molecular machines that utilize the energy of ATP turnover to generate force and perform various functions in eukaryotic cells. These enzymes coordinate movements of conserved structural elements located at the nucleotide binding site (P-loop, switch-1, switch-2) with structural elements that interact with the filament surface (actin-or microtubule-binding interface) (1)(2)(3)(4)(5)(6)(7)(8). The ATPase activity and enzymatic efficiency of these molecular motors are activated in the presence of their filament partner, which is thought to be mediated through acceleration of the rate of product release (reviewed in ref 9). However, the structural basis for this phenomenon is not well understood.The ATPase mechanisms of several different monomeric kinesins have been extensively studied in the presence of microtubules: conventional kinesin/Kinesin-1 (10-12), Eg5/ Kinesin-5 (13), and Ncd/Kar3/ . On the other hand, very little is known about the ATPase mechanism of kinesins in the absence of microtubules (17). Historically, kinesins have been purified with ADP bound to the nucleotide binding site (18), and attempts † This work was supported by Grant GM54141 from NIGMS, National Institutes of Health (NIH), and through Career Development Award K02-AR47841 from NIAMS, NIH, Department of Health and Human Services (to S.P.G.). to isolate a homogeneous, nucleotide-free population have been difficult due to the in...

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“…Three microliters of the reactions (performed in triplicate) were spotted onto a piece of nitrocellulose mounted onto a dot-blot apparatus (BioRad) with a piece of Immobilon Ny + below as described elsewhere (28,29). All reactions were performed at least in triplicate.…”

Section: Double Filter Binding Assaymentioning

confidence: 99%

“…To verify the existence of two different ATPase sites in Lon under our reaction conditions, we measured the affinities of Lon for α[ 32 P]ATP using a filter binding assay adapted from the protocols of Gilbert (27,28) and Lohman (29). The half life of the complex where ATP is bound at the low affinity sites can be calculated using the off rate of ATP (Vineyard and Lee manuscript in preparation).…”

Section: Examining Binding Of the Atpase Sites In Lonmentioning

confidence: 99%

Single-Turnover Kinetic Experiments Confirm the Existence of High- and Low-Affinity ATPase Sites in Escherichia coli Lon Protease

2006

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Lon is an ATP-dependent serine protease which degrades damaged and certain regulatory proteins in vivo. Lon exists as a homo-oligomer and represents one of the simplest ATP dependent proteases because both the protease and ATPase domains are located within each monomeric subunit. Previous pre-steady state kinetic studies revealed functional non-equivalency in the ATPase activity of the enzyme [Vineyard, D., et al. (2005) Biochemistry 44, 1671. Both a high and low affinity ATPase site has been previously reported for Lon [Menon, A.S., & Goldberg, A.L. (1987) J Biol Chem 262, 14921-8]. Because of the differing affinities for ATP, we were able to monitor the activities of the sites separately and determine that they were non-interacting. The high affinity sites hydrolyze ATP very slowly (k obs =0.019 ± 0.002s −1 ), while the low affinity sites hydrolyze ATP quickly at a rate of 17.2 ± 0.09s −1 which is comparable to the previously observed burst rate. Although the high affinity sites hydrolyze ATP slowly they support multiple rounds of peptide hydrolysis, indicating that ATP and peptide hydrolysis are not stoichiometrically linked. However, ATP binding and hydrolysis at both the high and low affinity sites is necessary for optimal peptide cleavage and the stabilization of the conformational change associated with nucleotide binding.Lon is an ATP dependent serine protease functioning to degrade damaged and certain regulatory proteins in vivo (1-10). Lon belongs to the ATPases associated with a variety of cellular activities (AAA + ) superfamily whose members include ClpAP, ClpXP, ClpCP, and HslVU (11,12). They share a conserved Walker A (or P-loop) and Walker B motif which is associated with nucleotide binding and hydrolysis (13). Lon represents one of the simplest of the ATP dependent proteases because both the protease and ATPase domains are located within each monomeric subunit (14,15). Crystal structures of portions of the enzyme have been recently reported, and include an inactive mutant of the Lon protease domain (16)(17)(18). This structure shows Lon as a hexamer organized in a ring with a central cavity which is commonly found in other ATP dependent proteases (11,13,17). Although it is known that ATP modulates Lon's protease activity (4,5,7,19), mechanistic details concerning how the binding and hydrolysis of ATP is coordinated with peptide bond cleavage is not known. However, it has been shown that ATP binding and hydrolysis does not affect the oligomeric state of the enzyme (20,21).We have previously developed a continuous fluorescent peptidase assay to monitor the kinetics of peptide cleavage. As the inner filter effect of fluorescence interferes at high concentrations of 100% fluorescent peptide, we used S3, a 10% mixture of fluorescently labeled peptide with its non-fluorescent analog (S2) (22). No optical signal from the peptide is needed when monitoring ATPase activity so only the non-fluorescent analog (S2) is used to account for the *Corresponding author, email: Irene.lee@case.edu, NIH Pub...

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Kinetics: A Tool to Study Molecular Motors

Cited by 69 publications

References 66 publications

Identification of the Proteasome Inhibitor MG262 as a Potent ATP-Dependent Inhibitor of the Salmonella enterica serovar Typhimurium Lon Protease

Identification of the Proteasome Inhibitor MG262 as a Potent ATP-Dependent Inhibitor of the Salmonella enterica serovar Typhimurium Lon Protease

ATPase Mechanism of Eg5 in the Absence of Microtubules: Insight into Microtubule Activation and Allosteric Inhibition by Monastrol

Single-Turnover Kinetic Experiments Confirm the Existence of High- and Low-Affinity ATPase Sites in Escherichia coli Lon Protease

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