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We demonstrate a method for label-free monitoring of hydrolytic activity of crystalline-chitin-degrading enzyme, chitinase, by means of Raman spectroscopy. We found that crystalline chitin exhibited a characteristic Raman peak at 2995 cm−1, which did not appear in the reaction product, N,N′-diacetylchitobiose. We used this Raman peak as a marker of crystalline chitin degradation to monitor the hydrolytic activity of chitinase. When the crystalline chitin suspension and chitinase were mixed together, the peak intensity of crystalline chitin at 2995 cm−1 was linearly decreased depending on incubation time. The decrease in peak intensity was inversely correlated with the increase in the amount of released N,N′-diacetylchitobiose, which was measured by conventional colorimetric assay with alkaline ferricyanide. Our result, presented here, provides a new method for simple, in situ, and label-free monitoring of enzymatic activity of chitinase. [ABSTRACT FROM AUTHOR]

A catalytically important arginine, called Arg finger, is employed in many enzymes to regulate their functions through enzymatic hydrolysis of nucleotide triphosphates. F1-ATPase (F1), a rotary motor protein, possesses Arg fingers which catalyze hydrolysis of adenosine triphosphate (ATP) for efficient chemomechanical energy conversion. In this study, we examined the Arg finger catalysis by single-molecule measurements for a mutant of F1 in which the Arg finger is substituted with an unnatural amino acid of a lysine analogue, 2,7-diaminoheptanoic acid (Lyk). The use of Lyk, of which the side chain is elongated by one CH2 unit so that its chain length to the terminal nitrogen of amine is set to be equal to that of arginine, allowed us to resolve key chemical factors in the Arg finger catalysis, i.e., chain length matching and chemical properties of the terminal groups. Rate measurements by single-molecule observations showed that the chain length matching of the side-chain length is not a sole requirement for the Arg finger to catalyze the ATP hydrolysis reaction step, indicating the crucial importance of chemical properties of the terminal guanidinium group in the Arg finger catalysis. On the other hand, the Lyk mutation prevented severe formation of an ADP inhibited state observed for a lysine mutant and even improved the avoidance of inhibition compared with the wild-type F1. The present study demonstrated that incorporation of unnatural amino acids can widely extend with its high "chemical" resolution biochemical approaches for elucidation of the molecular mechanism of protein functions and furnishing novel characteristics. [ABSTRACT FROM AUTHOR]

V1-ATPase is a rotary molecular motor in which the mechanical rotation of the rotor DF subunits against the stator A3B3 ring is driven by the chemical free energy of ATP hydrolysis. Recently, using X-ray crystallography, we solved the high-resolution molecular structure of Enterococcus hirae V1-ATPase (EhV1) and revealed how the three catalytic sites in the stator A3B3 ring change their structure on nucleotide binding and interaction with the rotor DF subunits. Furthermore, recently, we also demonstrated directly the rotary catalysis of EhV1 by using single-molecule high-speed imaging and analyzed the properties of the rotary motion in detail. In this critical review, we introduce the molecular structure and rotary dynamics of EhV1 and discuss a possible model of its chemomechanical coupling scheme. © 2014 IUBMB Life, 66(9):624-630, 2014 [ABSTRACT FROM AUTHOR]

Ring-shaped nucleoside triphosphatases (ring NTPases) are biological molecular machines powered by energy from NTP hydrolysis and are responsible for various cellular activities. These ring NTPases translocate their substrates or rotate their own subunits to/in the hole of the ring. Coordination and cooperativity among subunits in the oligomer ring is a topic of debate focused on understanding the operation mechanism of these protein machines. With the help of X-ray crystallographic structural analysis and optical microscopic single-molecules studies, distinct models, including stochastic, concerted, and rotary catalysis have been proposed. Here, we discuss these models and introduce high-speed atomic force microscopy as a new potent tool for verification of the model, with our recent example of the rotary catalysis of the stator ring of F1-adenosine triphosphatase. [Copyright &y& Elsevier]

Abstract: F1-ATPase is a rotary motor protein in which 3 catalytic β-subunits in a stator α3β3 ring undergo unidirectional and cooperative conformational changes to rotate the rotor γ-subunit upon adenosine triphosphate hydrolysis. The prevailing view of the mechanism behind this rotary catalysis elevated the γ-subunit as a “dictator” completely controlling the chemical and conformational states of the 3 catalytic β-subunits. However, our recent observations using high-speed atomic force microscopy clearly revealed that the 3 β-subunits undergo cyclic conformational changes even in the absence of the rotor γ-subunit, thus dethroning it from its dictatorial position. Here, we introduce our results in detail and discuss the possible operating principle behind the F1-ATPase, along with structurally related hexameric ATPases, also mentioning the possibility of generating hybrid nanomotors. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012). [Copyright &y& Elsevier]

The conformational fluctuation of enzymes has a crucial role in reaction acceleration. However, the contribution to catalysis enhancement of individual substates with conformations far from the average conformation remains unclear. We studied the catalytic power of the rotary molecular motor F1-ATPase from thermophilic Bacillus PS3 as it was stalled in transient conformations far from a stable pausing angle. The rate constants of ATP binding and hydrolysis were determined as functions of the rotary angle. Both rates exponentially increase with rotation, revealing the molecular basis of positive cooperativity among three catalytic sites: elementary reaction steps are accelerated via the mechanical rotation driven by other reactions on neighboring catalytic sites. The rate enhancement induced by ATP binding upon rotation was greater than that brought about by hydrolysis, suggesting that the ATP binding step contributes more to torque generation than does the hydrolysis step. Additionally, 9% of the ATP-driven rotary step was supported by thermal diffusion, suggesting that acceleration of the ATP docking process occurs via thermally agitated conformational fluctuations. [ABSTRACT FROM AUTHOR]

FoF1-ATP synthase is one of the most ubiquitous enzymes; it is found widely in the biological world, including the plasma membrane of bacteria, inner membrane of mitochondria and thylakoid membrane of chloroplasts. However, this enzyme has a unique mechanism of action: it is composed of two mechanical rotary motors, each driven by ATP hydrolysis or proton flux down the membrane potential of protons. The two molecular motors interconvert the chemical energy of ATP hydrolysis and proton electrochemical potential via the mechanical rotation of the rotary shaft. This unique energy transmission mechanism is not found in other biological systems. Although there are other similar man-made systems like hydroelectric generators, FoF1-ATP synthase operates on the nanometre scale and works with extremely high efficiency. Therefore, this enzyme has attracted significant attention in a wide variety of fields from bioenergetics and biophysics to chemistry, physics and nanoscience. This review summarizes the latest findings about the two motors of FoF1-ATP synthase as well as a brief historical background. [ABSTRACT FROM PUBLISHER]

F1-ATPase is an ATP-driven rotary motor protein in which the γ-subunit rotates against the catalytic stator ring. Although the reaction scheme of F1 has mostly been revealed, the timing of inorganic phosphate (Pi) release remains controversial. Here we addressed this issue by verifying the reversibility of ATP hydrolysis on arrested F1 with magnetic tweezers. ATP hydrolysis was found to be essentially reversible, implying that Pi is released after the γ rotation and ADP release, although extremely slow Pi release was found at the ATP hydrolysis angle as an uncoupling side reaction. On the basis of this finding, we deduced the chemomechanical coupling scheme of F1. We found that the affinity for Pi was strongly angle dependent, implying a large contribution by Pi release to torque generation. These findings imply that under ATP synthesis conditions, Pi binds to an empty catalytic site, preventing solution ATP (though not ADP) from binding. Thus, this supports the concept of selective ADP binding for efficient ATP synthesis. [ABSTRACT FROM AUTHOR]

F-ATPase is a molecular motor in which the γ subunit rotates inside the αβ ring upon adenosine triphosphate (ATP) hydrolysis. Recent works on single-molecule manipulation of F-ATPase have shown that kinetic parameters such as the on-rate of ATP and the off-rate of adenosine diphosphate (ADP) strongly depend on the rotary angle of the γ subunit (Hirono-Hara et al. ; Iko et al. ). These findings provide important insight into how individual reaction steps release energy to power F and also have implications regarding ATP synthesis and how reaction steps are reversed upon reverse rotation. An important issue regarding the angular dependence of kinetic parameters is that the angular position of a magnetic bead rotation probe could be larger than the actual position of the γ subunit due to the torsional elasticity of the system. In the present study, we assessed the stiffness of two different portions of F from thermophilic Bacillus PS3: the internal part of the γ subunit embedded in the αβ ring, and the complex of the external part of the γ subunit and the αβ ring (and streptavidin and magnetic bead), by comparing rotational fluctuations before and after crosslinkage between the rotor and stator. The torsional stiffnesses of the internal and remaining parts were determined to be around 223 and 73 pNnm/radian, respectively. Based on these values, it was estimated that the actual angular position of the internal part of the γ subunit is one-fourth of the magnetic bead position upon stalling using an external magnetic field. The estimated elasticity also partially explains the accommodation of the intrinsic step size mismatch between F and F-ATPase. [ABSTRACT FROM AUTHOR]

TheE subunit of bacterial F[sub0]F[sub1]-ATP synthase (F[sub0]F[sub1]), a rotary motor protein, is known to inhibit the ATP hydrolysis reaction of this enzyme. The inhibitory effect is modulated by the conformation of the C-terminal α-helices of ϵ, and the "extended" but not "hairpin-folded" state is responsible for inhibition. Although the inhibition of ATP hydrolysis by the C-terminal domain of ϵ has been extensively studied, the effect on ATP synthesis is not fully understood. In this study, we generated an Escherichia coli F[sub0]F[sub1] (EF[sub0]F[sub1]) mutant in which the &3x20AC; subunit lacked the C-terminal domain (F[sub0]F[sub1][supϵΔC]), and ATP synthesis driven by acid-base transition (ΔpH) and the K[sup+]-valinomycin diffusion potential (ΔΨ) was compared in detail with that of the wild-type enzyme (F[sub0]F[sub1][supϵWT]). The turnover numbers (kcat) of F[sub0]F[sub1][supϵWT] were severalfold lower than those of F[sub0]F[sub1][supϵΔC]. F[sub0]F[sub1][supϵWT] showed higher Michaelis constants (K,,). The dependence of the activities of F[sub0]F[sub1][supϵWT] and F[sub0]F[sub1][supϵΔC] on various combinations of ΔpH and ΔΨ was similar, suggesting that the rate-limiting step in ATP synthesis was unaltered by the C-terminal domain of ϵ. Solubilized F[sub0]F[sub1][supϵWT] also showed lower kcat and higher Km values for ATP hydrolysis than the corresponding values of F[sub0]F[sub1][supϵΔC]. These results suggest that the C-terminal domain of the ϵ subunit of EF[sub0]F[sub1] slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the γ subunit. [ABSTRACT FROM AUTHOR]

F1-ATPase is a rotary molecular motor driven by ATP hydrolysis that rotates the γ-subunit against the α3β3 ring. The crystal structures of F1, which provide the structural basis for the catalysis mechanism, have shown essentially 1 stable conformational state. In contrast, single-molecule studies have revealed that F1 has 2 stable conformational states: ATP-binding dwell state and catalytic dwell state. Although structural and single-molecule studies are crucial for the understanding of the molecular mechanism of F1, it remains unclear as to which catalytic state the crystal structure represents. To address this issue, we introduced cysteine residues at βE391 and γR84 of F1 from thermophilic Bacillus PS3. In the crystal structures of the mitochondrial F1, the corresponding residues in the ADP-bound β (βDP) and γ were in direct contact. The βE190D mutation was additionally introduced into the β to slow ATP hydrolysis. By incorporating a single copy of the mutant β-subunit, the chimera F1, α3β2β(E190D/E391 C)β(R84C), was prepared. In single-molecule rotation assay, chimera F1 showed a catalytic dwell pause in every turn because of the slowed ATP hydrolysis of β(E190D/E391C). When the mutant β and γ were cross-linked through a disulfide bond between βE391C and γR84C, F1 paused the rotation at the catalytic dwell angle of β(E190D/E391C), indicating that the crystal structure represents the catalytic dwell state and that βDP is the catalytically active form. The former point was again confirmed in experiments where F1 rotation was inhibited by adenosine-5'- (β,γ-imino)-triphosphate and/or azide, the most commonly used inhibitors for the crystallization of F1. [ABSTRACT FROM AUTHOR]

It has been proposed that C-terminal two α-helices of the ∈ subunit of F1-ATPase can undergo conformational transition between retracted folded-hairpin form and extended form. Here, using F1 from thermophilic Bacillus PS3, we monitored this transition in real time by fluorescence resonance energy transfer (FRET) between a donor dye and an acceptor dye attached to N terminus of the γ subunit and C terminus of the β subunit, respectively. High FRET (extended form) of F1 turned to low FRET (retracted form) by ATP, which then reverted as ATP was hydrolyzed to ADP. 5′-Adenyl-β,γ-imidodiphosphate, ADP + AlF4-, ADP + NaN3, and GTP also caused the retracted form, indicating that ATP binding to the catalytic/3 subunits induces the transition. The ATP-induced transition from high FRET to low FRET occurred in a similar time scale to the ATP-induced activation of ATPase from inhibition by the β subunit, although detailed kinetics were not the same. The transition became faster as temperature increased, but the extrapolated rate at 65 °C (physiological temperature of Bacillus PS3) was still too slow to assign the transition as an obligate step in the catalytic turnover. Furthermore, binding affinity of ATP to the isolated subunit was weakened as temperature increased, and the dissociation constant extrapolated to 65 °C reached to 0.67 mM, a consistent value to assume that the ∈ subunit acts as a sensor of ATP concentration in the cell. [ABSTRACT FROM AUTHOR]

F1-ATPase (F1) is an ATP-driven rotary motor wherein the γ subunit rotates against the surrounding α3β3 stator ring. The 3 catalytic sites of F1 reside on the interface of the α and β subunits of the α3β3 ring. While the catalytic residues predominantly reside on the β subunit, the α subunit has 1 catalytically critical arginine, termed the arginine finger, with stereogeometric similarities with the arginine finger of G-protein-activating proteins. However, the principal role of the arginine finger of F1 remains controversial.Westudied the role of the arginine finger by analyzing the rotation of a mutant F1 with a lysine substitution of the arginine finger. The mutant showed a 350-fold longer catalytic pause than the wild-type; this pause was further lengthened by the slowly hydrolyzed ATP analog ATPγS. On the other hand, the mutant F1 showed highly unidirectional rotation with a coupling ratio of 3 ATPs/turn, the same as wild-type, suggesting that cooperative torque generation by the 3 β subunits was not impaired. The hybrid F1 carrying a single copy of the β mutant revealed that the reaction step slowed by the mutation occurs at +200° from the binding angle of the mutant subunit. Thus, the principal role of the arginine finger is not to mediate cooperativity among the catalytic sites, but to enhance the rate of the ATP cleavage by stabilizing the transition state of ATP hydrolysis. Lysine substitution also caused frequent pauses because of severe ADP inhibition, and a slight decrease in ATPbinding rate. [ABSTRACT FROM AUTHOR]

Enzymatic hydrolysis of nucleotide triphosphate (NTP) plays a pivotal role in protein functions. In spite of its biological significance, however, the chemistry of the hydrolysis catalysis remains obscure because of the complex nature of the reaction. Here we report a study of the molecular mechanism of hydrolysis of adenosine triphosphate (ATP) in F1-ATPase, an ATP-driven rotary motor protein. Molecular simulations predicted and single-molecule observation experiments verified that the rate-determining step (RDS) is proton transfer (PT) from the lytic water molecule, which is strongly activated by a metaphosphate generated by a preceding Pγ-Oβ bond dissociation (POD). Catalysis of the POD that triggers the chain activation of the PT is fulfilled by hydrogen bonds between Walker motif A and an arginine finger, which commonly exist in many NTPases. The reaction mechanism unveiled here indicates that the protein can regulate the enzymatic activity for the function in both the POD and PT steps despite the fact that the RDS is the PT step. [ABSTRACT FROM AUTHOR]