Single Turnover Conditions Research Articles

Kinetic Characterization of the ATPase and Actin-activated ATPase Activities of Acanthamoeba castellanii Myosin-2

Phosphorylation of Ser-639 in loop-2 of the catalytic motor domain of the heavy chain of Acanthamoeba castellanii myosin-2 and the phosphomimetic mutation S639D have been shown previously to down-regulate the actin-activated ATPase activity of both the full-length myosin and single-headed subfragment-1 (Liu, X., Lee, D. Y., Cai, S., Yu, S., Shu, S., Levine, R. L., and Korn, E. D. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, E23-E32). In the present study we determined the kinetic constants for each step in the myosin and actomyosin ATPase cycles of recombinant wild-type S1 and S1-S639D. The kinetic parameter predominantly affected by the S639D mutation is the actin-activated release of inorganic phosphate from the acto myosin·ADP·Pi complex, which is the rate-limiting step in the steady-state actomyosin ATPase cycle. As consequence of this change, the duty ratio of this conventional myosin decreases. We speculate on the effect of Ser-639 phosphorylation on the processive behavior of myosin-2 filaments.

Mechanistic insights into small RNA recognition and modification by the HEN1 methyltransferase

The HEN1 methyltransferase from Arabidopsis thaliana modifies the 3'-terminal nucleotides of small regulatory RNAs. Although it is one of the best characterized members of the 2'-O-methyltransferase family, many aspects of its interactions with the cofactor and substrate RNA remained unresolved. To better understand the substrate interactions and contributions of individual steps during HEN1 catalysis, we studied the binding and methylation kinetics of the enzyme using a series of unmethylated, hemimethylated and doubly methylated miRNA and siRNA substrates. The present study shows that HEN1 specifically binds double-stranded unmethylated or hemimethylated miR173/miR173* substrates with a subnanomolar affinity in a cofactor-dependent manner. Kinetic studies under single turnover and pre-steady state conditions in combination with isotope partitioning analysis showed that the binary HEN1-miRNA/miRNA* complex is catalytically competent; however, successive methylation of the two strands in a RNA duplex occurs in a non-processive (distributive) manner. We also find that the observed moderate methylation strand preference is largely exerted at the RNA-binding step and is fairly independent of the nature of the 3'-terminal nucleobase, but shows some dependency on proximal nucleotide mispairs. The results of the present study thus provide novel insights into the mechanism of RNA recognition and modification by a representative small RNA 2'-O-methyltransferase.

The tRNA-modifying function of MnmE is controlled by post-hydrolysis steps of its GTPase cycle

MnmE is a homodimeric multi-domain GTPase involved in tRNA modification. This protein differs from Ras-like GTPases in its low affinity for guanine nucleotides and mechanism of activation, which occurs by a cis, nucleotide- and potassium-dependent dimerization of its G-domains. Moreover, MnmE requires GTP hydrolysis to be functionally active. However, how GTP hydrolysis drives tRNA modification and how the MnmE GTPase cycle is regulated remains unresolved. Here, the kinetics of the MnmE GTPase cycle was studied under single-turnover conditions using stopped- and quench-flow techniques. We found that the G-domain dissociation is the rate-limiting step of the overall reaction. Mutational analysis and fast kinetics assays revealed that GTP hydrolysis, G-domain dissociation and Pi release can be uncoupled and that G-domain dissociation is directly responsible for the ‘ON’ state of MnmE. Thus, MnmE provides a new paradigm of how the ON/OFF cycling of GTPases may regulate a cellular process. We also demonstrate that the MnmE GTPase cycle is negatively controlled by the reaction products GDP and Pi. This feedback mechanism may prevent inefficacious GTP hydrolysis in vivo. We propose a biological model whereby a conformational change triggered by tRNA binding is required to remove product inhibition and initiate a new GTPase/tRNA-modification cycle.

Switching from single-stranded to double-stranded DNA limits the unwinding processivity of ring-shaped T7 DNA helicase

Phage T7 helicase unwinds double-stranded DNA (dsDNA) by encircling one strand while excluding the complementary strand from its central channel. When T7 helicase translocates on single-stranded DNA (ssDNA), it has kilobase processivity; yet, it is unable to processively unwind linear dsDNA, even 60 base-pairs long. Particularly, the GC-rich dsDNAs are unwound with lower amplitudes under single-turnover conditions. Here, we provide evidence that T7 helicase switches from ssDNA to dsDNA during DNA unwinding. The switching propensity is higher when dsDNA is GC-rich or when the 3′-overhang of forked DNA is <15 bases. Once helicase encircles dsDNA, it travels along dsDNA and dissociates from the end of linear DNA without strand separation, which explains the low unwinding amplitude of these substrates. Trapping the displaced strand with ssDNA binding protein or changing its composition to morpholino oligomer that does not interact with helicase increases the unwinding amplitude. We conclude that the displaced strand must be continuously excluded and kept away from the central channel for processive DNA unwinding. The finding that T7 helicase can switch from ssDNA to dsDNA binding mode during unwinding provides new insights into ways of limiting DNA unwinding and triggering fork regression when stalled forks need to be restarted.

TRNA residues evolved to promote translational accuracy

The decoding properties of 22 structurally conservative base-pair and base-triple mutations in the anticodon hairpin and tertiary core of Escherichia coli tRNA(Ala)GGC were determined under single turnover conditions using E. coli ribosomes. While all of the mutations were able to efficiently decode the cognate GCC codon, many showed substantial misreading of near-cognate GUC or ACC codons. Although all the misreading mutations were present in the sequences of other E. coli tRNAs, they were never found among bacterial tRNA(Ala)GGC sequences. This suggests that the sequences of bacterial tRNA(Ala)GGC have evolved to avoid reading incorrect codons.

Transient Kinetic Analysis of USP2-Catalyzed Deubiquitination Reveals a Conformational Rearrangement in the K48-Linked Diubiquitin Substrate

Deubiquitination has emerged as an essential regulatory mechanism of a number of cellular processes. An in-depth understanding of deubiquitinating enzyme (DUB) catalysis, particularly the mode of ubiquitin binding and the individual steps in the DUB catalytic turnover, is imperative for exploiting DUBs for therapeutic intervention. In this work, we present a transient kinetic study of USP2 in hydrolyzing a model substrate Ub-AMC and a physiological substrate K48-linked diubiquitin. We conducted stopped-flow fluorescence analyses of the binding of mono- and diubiquitin to an inactive USP2 mutant and unveiled interesting differences in the binding kinetics between the two substrates. While a simple one-step binding of monoubiquitin to USP2 was observed, a biphasic binding was evident for diubiquitin. We further followed the deubiquitination reaction of Ub-AMC and K48-linked IQF-diubiquitin by USP2 using stopped-flow florescence under a single-turnover condition. Global fitting of the reaction traces revealed differences in the microscopic rate constants between Ub-AMC and the physiological diubiquitin substrate. Our binding and single-turnover data support a conformational rearrangement of the diubiquitin substrate in USP2-catalyzed deubiquitination. This finding is significant given the recent finding that the K48-linked diubiquitin is dynamic in its conformation. Our results provide useful insights into the mechanism of how USP recognizes ubiquitin moieties in a chain structure, which is important for understanding USP catalysis and developing inhibitors against USPs.

Evidence for a dual functional role of a conserved histidine in RNA·DNA heteroduplex cleavage by human RNase H1

Ribonuclease H1 is a conserved enzyme that cleaves the RNA strand of RNA·DNA heteroduplexes and has important functions in the nuclear and mitochondrial compartments. The therapeutic action of antisense oligodeoxynucleotides involves the recruitment of RNase H1 to cleave disease-relevant RNA targets. Recombinant human (Hs) RNase H1 was purified from a bacterial expression host, and conditions were identified that provided optimal oligonucleotide-directed RNA cleavage invitro. Hs-RNase H1 exhibits optimal catalytic activity in pH 7.5 HEPES buffer and a salt (KCl) concentration of ~100-150mm. Mg(2+) best supports Hs-RNase H1 with an optimal concentration of 10mm, but at higher concentrations inhibits enzyme activity. Mn(2+) and Co(2+) also support catalytic activity, while Ni(2+) and Zn(2+) exhibit only modest activities as cofactors. The optimized assay was used to show that an antisense oligonucleotide, added in substoichiometric amounts to initiate RNA cleavage, supports up to 30 rounds of reaction in 30min. Mutation to alanine of the conserved histidine at position 264 causes an ~100-fold decrease in k(cat) under multiple-turnover conditions, but does not alter K(m) . Under single-turnover conditions, the H264A mutant exhibits a 12-fold higher exponential time constant for substrate cleavage. The defective activity of the H264A mutant is not rescued in either assay condition by higher Mg(2+) concentrations. These data implicate the H264 side chain in phosphodiester hydrolysis as well as in product release, and are consistent with a proposed model in which the H264 side chain interacts with a divalent metal ion to support catalysis.

Two Pyrenylalanines in Dihydrofolate Reductase Form an Excimer Enabling the Study of Protein Dynamics

Because of the lack of sensitivity to small changes in distance by available FRET pairs (a constraint imposed by the dimensions of the enzyme), a DHFR containing two pyrene moieties was prepared to enable the observation of excimer formation. Pyren-1-ylalanine was introduced into DHFR positions 16 and 49 using an in vitro expression system in the presence of pyren-1-ylalanyl-tRNA(CUA). Excimer formation (λ(ex) 342 nm; λ(em) 481 nm) was observed in the modified DHFR, which retained its catalytic competence and was studied under multiple and single turnover conditions. The excimer appeared to follow a protein conformational change after the H transfer involving the relative position and orientation of the pyrene moieties and is likely associated with product dissociation.

Catalytic Electron‐Transfer Oxygenation of Substrates with Water as an Oxygen Source Using Manganese Porphyrins

Manganese(V)-oxo-porphyrins are produced by the electron-transfer oxidation of manganese-porphyrins with tris(2,2'-bipyridine)ruthenium(III) ([Ru(bpy)(3)](3+); 2 equiv) in acetonitrile (CH(3)CN) containing water. The rate constants of the electron-transfer oxidation of manganese-porphyrins have been determined and evaluated in light of the Marcus theory of electron transfer. Addition of [Ru(bpy)(3)](3+) to a solution of olefins (styrene and cyclohexene) in CH(3)CN containing water in the presence of a catalytic amount of manganese-porphyrins afforded epoxides, diols, and aldehydes efficiently. Epoxides were converted to the corresponding diols by hydrolysis, and were further oxidized to the corresponding aldehydes. The turnover numbers vary significantly depending on the type of manganese-porphyrin used owing to the difference in their oxidation potentials and the steric bulkiness of the ligand. Ethylbenzene was also oxidized to 1-phenylethanol using manganese-porphyrins as electron-transfer catalysts. The oxygen source in the substrate oxygenation was confirmed to be water by using (18)O-labeled water. The rate constant of the reaction of the manganese(V)-oxo species with cyclohexene was determined directly under single-turnover conditions by monitoring the increase in absorbance attributable to the manganese(III) species produced in the reaction with cyclohexene. It has been shown that the rate-determining step in the catalytic electron-transfer oxygenation of cyclohexene is electron transfer from [Ru(bpy)(3)](3+) to the manganese-porphyrins.

Structure and Dynamics of the ATP-Bound Open Conformation of Hsp70 Chaperones

Central to the chaperone function of Hsp70s is the transition between open and closed conformations of their polypeptide substrate binding domain (SBD), which is regulated through an allosteric mechanism via ATP binding and hydrolysis in their nucleotide binding domain (NBD). Although the structure of the closed conformation of Hsp70s is well studied, the open conformation has remained elusive. Here, we report on the 2.4Å crystal structure of the ATP-bound open conformation of the Escherichia coli Hsp70 homolog DnaK. In the open DnaK structure, the β sheet and α-helical lid subdomains of the SBD are detached from one another and docked to different faces of the NBD. The contacts between the β sheet subdomain and the NBD reveal the mechanism of allosteric regulation. In addition, we demonstrate that docking of the β sheet and α-helical lid subdomains to the NBD is a sequential process influenced by peptide and protein substrates.

Single turnover studies of oxidative halophenol dehalogenation by horseradish peroxidase reveal a mechanism involving two consecutive one electron steps: Toward a functional halophenol bioremediation catalyst

Horseradish peroxidase (HRP) catalyzes the oxidative para-dechlorination of the environmental pollutant/carcinogen 2,4,6-trichlorophenol (2,4,6-TCP). A possible mechanism for this reaction is a direct oxygen atom transfer from HRP compound I (HRP I) to trichlorophenol to generate 2,6-dichloro 1,4-benzoquinone, a two-electron transfer process. An alternative mechanism involves two consecutive one-electron transfer steps in which HRP I is reduced to compound II (HRP II) and then to the ferric enzyme as first proposed by Wiese et al. [F.W. Wiese, H.C. Chang, R.V. Lloyd, J.P. Freeman, V.M. Samokyszyn, Arch. Environ. Contam. Toxicol. 34 (1998) 217–222]. To probe the mechanism of oxidative halophenol dehalogenation, the reactions between 2,4,6-TCP and HRP compounds I or II have been investigated under single turnover conditions (i.e., without excess H2O2) using rapid scan stopped-flow spectroscopy. Addition of 2,4,6-TCP to HRP I leads rapidly to HRP II and then more slowly to the ferric resting state, consistent with a mechanism involving two consecutive one-electron oxidations of the substrate via a phenoxy radical intermediate. HRP II can also directly dechlorinate 2,4,6-TCP as judged by rapid scan stopped-flow and mass spectrometry. This observation is particularly significant since HRP II can only carry out one-electron oxidations. A more detailed understanding of the mechanism of oxidative halophenol dehalogenation will facilitate the use of HRP as a halophenol bioremediation catalyst.

TRNA Processing by Protein‐Only versus RNA‐Based RNase P: Kinetic Analysis Reveals Mechanistic Differences

In Arabidopsis thaliana, RNase P function, that is, endonucleolytic tRNA 5'-end maturation, is carried out by three homologous polypeptides ("proteinaceous RNase P" (PRORP) 1, 2 and 3). Here we present the first kinetic analysis of these enzymes. For PRORP1, a specificity constant (k(react)/K(m(sto))) of 3×10(6) M(-1) min(-1) was determined under single-turnover conditions. We demonstrate a fundamentally different sensitivity of PRORP enzymes to an Rp-phosphorothioate modification at the canonical cleavage site in a 5'-precursor tRNA substrate; whereas processing by bacterial RNase P is inhibited by three orders of magnitude in the presence of this sulfur substitution and Mg(2+) as the metal-ion cofactor, the PRORP enzymes are affected by not more than a factor of five under the same conditions, without significantly increased miscleavage. These findings indicate that the catalytic mechanism utilized by proteinaceous RNase P is different from that of RNA-based bacterial RNase P, taking place without a direct metal-ion coordination to the (pro-)Rp substituent. As Rp-phosphorothioate and inosine modification at all 26 G residues of the tRNA body had only minor effects on processing by PRORP, we conclude that productive PRORP-substrate interaction is not critically dependent on any of the affected (pro-)Rp oxygens or guanosine 2-amino groups.

Charge Compensation Mechanism of a Na+-coupled, Secondary Active Glutamate Transporter

Forward glutamate transport by the excitatory amino acid carrier EAAC1 is coupled to the inward movement of three Na(+) and one proton and the subsequent outward movement of one K(+) in a separate step. Based on indirect evidence, it was speculated that the cation binding sites bear a negative charge. However, little is known about the electrostatics of the transport process. Valences calculated using the Poisson-Boltzmann equation indicate that negative charge is transferred across the membrane when only one cation is bound. Consistently, transient currents were observed in response to voltage jumps when K(+) was the only cation on both sides of the membrane. Furthermore, rapid extracellular K(+) application to EAAC1 under single turnover conditions (K(+) inside) resulted in outward transient current. We propose a charge compensation mechanism, in which the C-terminal transport domain bears an overall negative charge of -1.23. Charge compensation, together with distribution of charge movement over many steps in the transport cycle, as well as defocusing of the membrane electric field, may be combined strategies used by Na(+)-coupled transporters to avoid prohibitive activation barriers for charge translocation.

Temperature dependence of accuracy of DNA polymerase I from Geobacillus anatolicus

Klenow-like DNA polymerase I fragment from Geobacillus anatolicus (GF) was cloned and purified. The accuracy of GF was measured invitro at three different temperatures under single turnover conditions as well as using a forward mutation assay. In pre-steady-state kinetic measurements, when temperature was raised from 22°C to 50°C, the rate (k(pol)) for cognate dTTP and non-cognate dATP nucleotide incorporations increased six- and four-fold, respectively, whereas the K(d) for both nucleotide incorporations changed only slightly. As a result, the error frequency was remained constant (∼4×10(-4)) over this temperature range. The accuracy of GF was also measured using a forward mutation assay during a single cycle of DNA synthesis of the lacZα complementation gene in M13mp2 DNA. In this assay, which scores various types of replication errors, mutant frequency of GF was 5×10(-3) at 72°C which is four-fold higher than that of 37°C.

Hydroxylation of Aromatics with the Help of a Non‐Haem FeOOH: A Mechanistic Study under Single‐Turnover and Catalytic Conditions

Ferric-hydroperoxo complexes have been identified as intermediates in the catalytic cycle of biological oxidants, but their role as key oxidants is still a matter of debate. Among the numerous synthetic low-spin Fe(III)(OOH) complexes characterized to date, [(L(5)(2))Fe(OOH)](2+) is the only one that has been isolated in the solid state at low temperature, which has provided a unique opportunity for inspecting its oxidizing properties under single-turnover conditions. In this report we show that [(L(5)(2))Fe(OOH)](2+) decays in the presence of aromatic substrates, such as anisole and benzene in acetonitrile, with first-order kinetics. In addition, the phenol products are formed from the aromatic substrates with similar first-order rate constants. Combining the kinetic data obtained at different temperatures and under different single-turnover experimental conditions with experiments performed under catalytic conditions by using the substrate [1,3,5-D(3)]benzene, which showed normal kinetic isotope effects (KIE>1) and a notable hydride shift (NIH shift), has allowed us to clarify the role played by Fe(III)(OOH) in aromatic oxidation. Several lines of experimental evidence in support of the previously postulated mechanism for the formation of two caged Fe(IV)(O) and OH(·) species from the Fe(III)(OOH) complex have been obtained for the first time. After homolytic O-O cleavage, a caged pair of oxidants [Fe(IV)O+HO(·)] is generated that act in unison to hydroxylate the aromatic ring: HO(·) attacks the ring to give a hydroxycyclohexadienyl radical, which is further oxidized by Fe(IV)O to give a cationic intermediate that gives rise to a NIH shift upon ketonization before the final re-aromatization step. Spin-trapping experiments in the presence of 5,5-dimethyl-1-pyrroline N-oxide and GC-MS analyses of the intermediate products further support the proposed mechanism.

Charge Compensation Mechanism of Glutamate Transport

Forward glutamate transport by the excitatory amino acid carrier EAAC1 is coupled to the inward movement of three Na+ and one proton, and the outward movement of one K+. Glutamate, Na+ and H+ are moved to the cytosol in the translocation steps. Subsequent outward movement of K+ occurs in a separate step. Based on indirect evidence, it was suggested that outward movement of positively-charged K+ is overcompensated by the outward movement of negative charge of the binding site(s). However, no information is available on the K+-dependent reaction step(s). Here, we examined the electrostatics of the glutamate transport process by evaluating implicit membrane/solvent/ explicit transporter models using the Poisson-Boltzmann (PB) equation. The results indicate that structural changes of the transporter bound to only one cation lead to transfer of negative charge across the membrane. In order to compensate for the negatively-charged binding sites, at least two Na+ and one proton need to be bound to the transporter. Consistent with these predictions, transient currents were observed in response to steps of the transmembrane potential when K+ was the only cation present on both sides of the membrane. No K+-dependent charge movements were observed in a transporter with the mutation E373Q, which is known to be defective in the K+-dependent relocation step(s), but not K+ binding. Furthermore, rapid extracellular K+ application to EAAC1 under single turnover conditions (only K+ inside) results in outward transient current, in contrast to the expected inward current in the absence of charge overcompensation. We propose a charge compensation mechanism for the transport process, in which the C-terminal transport domain is overall negatively charged, with an apparent valence of −1.26. Our model can be used to predict the kinetics of the K+-dependent half-cycle of the glutamate transport process.

Radical formation in cytochrome c oxidase

The formation of radicals in bovine cytochrome c oxidase (bC cO), during the O 2 redox chemistry and proton translocation, is an unresolved controversial issue. To determine if radicals are formed in the catalytic reaction of bC cO under single turnover conditions, the reaction of O 2 with the enzyme, reduced by either ascorbate or dithionite, was initiated in a custom-built rapid freeze quenching (RFQ) device and the products were trapped at 77 K at reaction times ranging from 50 μs to 6 ms. Additional samples were hand mixed to attain multiple turnover conditions and quenched with a reaction time of minutes. X-band (9 GHz) continuous wave electron paramagnetic resonance (CW-EPR) spectra of the reaction products revealed the formation of a narrow radical with both reductants. D-band (130 GHz) pulsed EPR spectra allowed for the determination of the g-tensor principal values and revealed that when ascorbate was used as the reductant the dominant radical species was localized on the ascorbyl moiety, and when dithionite was used as the reductant the radical was the SO 2 − ion. When the contributions from the reductants are subtracted from the spectra, no evidence for a protein-based radical could be found in the reaction of O 2 with reduced bC cO. As a surrogate for radicals formed on reaction intermediates, the reaction of hydrogen peroxide (H 2O 2) with oxidized bC cO was studied at pH 6 and pH 8 by trapping the products at 50 μs with the RFQ device to determine the initial reaction events. For comparison, radicals formed after several minutes of incubation were also examined, and X-band and D-band analysis led to the identification of radicals on Tyr-244 and Tyr-129. In the RFQ measurements, a peroxyl (R O O ) species was formed, presumably by the reaction between O 2 and an amino acid-based radical. It is postulated that Tyr-129 may play a central role as a proton loading site during proton translocation by ejecting a proton upon formation of the radical species and then becoming reprotonated during its reduction via a chain of three water molecules originating from the region of the propionate groups of heme a 3. This article is part of a Special Issue entitled: “Allosteric cooperativity in respiratory proteins”.

Kinetic Mechanism for the Excision of Hypoxanthine by Escherichia coli AlkA and Evidence for Binding to DNA Ends

The Escherichia coli 3-methyladenine DNA glycosylase II protein (AlkA) recognizes a broad range of oxidized and alkylated base lesions and catalyzes the hydrolysis of the N-glycosidic bond to initiate the base excision repair pathway. Although the enzyme was one of the first DNA repair glycosylases to be discovered more than 25 years ago and there are multiple crystal structures, the mechanism is poorly understood. Therefore, we have characterized the kinetic mechanism for the AlkA-catalyzed excision of the deaminated purine, hypoxanthine. The multiple-turnover glycosylase assays are consistent with Michaelis-Menten kinetics. However, under single-turnover conditions that are commonly employed for studying other DNA glycosylases, we observe an unusual biphasic protein saturation curve. Initially, the observed rate constant for excision increases with an increasing level of AlkA protein, but at higher protein concentrations, the rate constant decreases. This behavior can be most easily explained by tight binding to DNA ends and by crowding of multiple AlkA protamers on the DNA. Consistent with this model, crystal structures have shown the preferential binding of AlkA to DNA ends. By varying the position of the lesion, we identified an asymmetric substrate that does not show inhibition at higher concentrations of AlkA, and we performed pre-steady state and steady state kinetic analysis. Unlike the situation in other glycosylases, release of the abasic product is faster than N-glycosidic bond cleavage. Nevertheless, AlkA exhibits significant product inhibition under multiple-turnover conditions, and it binds approximately 10-fold more tightly to an abasic site than to a hypoxanthine lesion site. This tight binding could help protect abasic sites when the adaptive response to DNA alkylation is activated and very high levels of AlkA protein are present.

Mechanism of Resistance to GS-9148 Conferred by the Q151L Mutation in HIV-1 Reverse Transcriptase

GS-9148 is an investigational phosphonate nucleotide analogue inhibitor of reverse transcriptase (RT) (NtRTI) of human immunodeficiency virus type 1 (HIV-1). This compound is an adenosine derivative with a 2',3'-dihydrofuran ring structure that contains a 2'-fluoro group. The resistance profile of GS-9148 is unique in that the inhibitor can select for the very rare Q151L mutation in HIV-1 RT as a pathway to resistance. Q151L is not stably selected by any of the approved nucleoside or nucleotide analogues; however, it may be a transient intermediate that leads to the related Q151M mutation, which confers resistance to multiple compounds that belong to this class of RT inhibitors. Here, we employed pre-steady-state kinetics to study the impact of Q151L on substrate and inhibitor binding and the catalytic rate of incorporation. Most importantly, we found that the Q151L mutant is unable to incorporate GS-9148 under single-turnover conditions. Interference experiments showed that the presence of GS-9148-diphosphate, i.e., the active form of the inhibitor, does not reduce the efficiency of incorporation for the natural counterpart. We therefore conclude that Q151L severely compromises binding of GS-9148-diphosphate to RT. This effect is highly specific, since we also demonstrate that another NtRTI, tenofovir, is incorporated with selectivity similar to that seen with wild-type RT. Incorporation assays with other related compounds and models based on the RT/DNA/GS-9148-diphosphate crystal structure suggest that the 2'-fluoro group of GS-9148 may cause steric hindrance with the side chain of the Q151L mutant.

An Examination of the Kinetic Mechanism of the ATP-Dependent Protease ClpAP

Motor proteins perform functions within the cell by converting the energy of ATP into mechanical work. One such example of this can be found in the ATP-dependent protease from Escherichia coli, ClpAP, which is assembled from two distinct enzymes, a protein unfoldase, ClpA, and a protease, ClpP. The biologically active complex functions through a coordinated action in which ClpA is responsible for enzyme catalyzed protein unfolding and ATP-dependent polypeptide translocation, while ClpP will proteolytically degrade polypeptide substrates that have been translocated into its central cavity. Without such systems, deleterious effects are often observed within the cell as a result of either the accumulation of misfolded proteins or from unregulated activity from partially synthesized proteins.Of interest is the kinetic mechanism of ClpAP, and more specifically, the possibility of allosteric effects of ClpP upon ClpA. Kinetic parameters such as the step-size of polypeptide translocation, processvity, macroscopic rate, and microscopic rate constants have been measured for ClpA in the absence of ClpP under single-turnover conditions, but quantitative estimates of these parameters have not been determined in the presence of the proteolytic component, ClpP. In an effort to address this issue, single-turnover chemical quenched-flow methods in conjunction with stopped-flow fluorescence techniques have been used to examine the molecular mechanism of ClpAP catalyzed polypeptide translocation and degradation. The single-turnover methods are performed with enzyme in large excess over substrates and allow for the observation of the conversion of substrates into intermediates or products in a single cycle of catalysis. We have shown that the kinetic parameters are dramatically different in the presence of the proteolytic component and ClpP has a clear allosteric effect on the mechanism.