adenosine-5--o-(3-thiotriphosphate) and aluminum-fluoride

Aluminum fluoride has become an important tool for investigating the mechanism of phosphoryl transfer, an essential reaction that controls a host of vital cell functions. Planar AlF(3) or AlF(4)(-) molecules are proposed to mimic the phosphoryl group in the catalytic transition state. Acetate kinase catalyzes phosphoryl transfer of the ATP gamma-phosphate to acetate. Here we describe the inhibition of acetate kinase from Methanosarcina thermophila by preincubation with MgCl(2), ADP, AlCl(3), NaF, and acetate. Preincubation with butyrate in place of acetate did not significantly inhibit the enzyme. Several NTPs can substitute for ATP in the reaction, and the corresponding NDPs, in conjunction with MgCl(2), AlCl(3), NaF, and acetate, inhibit acetate kinase activity. Fluorescence quenching experiments indicated an increase in binding affinity of acetate kinase for MgADP in the presence of AlCl(3), NaF, and acetate. These and other characteristics of the inhibition indicate that the transition state analog, MgADP-aluminum fluoride-acetate, forms an abortive complex in the active site. The protection from inhibition by a non-hydrolyzable ATP analog or acetylphosphate, in conjunction with the strict dependence of inhibition on the presence of both ADP and acetate, supports a direct in-line mechanism for acetate kinase.

Topics: Acetate Kinase; Adenosine Diphosphate; Adenosine Triphosphate; Aluminum Compounds; Binding Sites; Butyrates; Dose-Response Relationship, Drug; Enzyme Inhibitors; Escherichia coli; Fluorides; Kinetics; Magnesium Chloride; Methanosarcina; Models, Chemical; Organophosphates; Protein Binding; Sodium Fluoride; Spectrometry, Fluorescence; Substrate Specificity; Time Factors

Motor proteins move unidirectionally along cytoskeletal polymers by coupling translocation to cycles of ATP hydrolysis. The energy from ATP is required both to generate force and to dissociate the motor-filament complex in order to begin a new chemomechanical cycle. For myosin, force production is associated with phosphate release following ATP hydrolysis, whereas dissociation of actomyosin is tightly coupled to the binding of ATP. Dynein, a microtubule motor, uses a similar cycle, suggesting that all cytoskeletal motors might operate by a common mechanism. Here we investigate kinesin's chemomechanical cycle by assaying microtubule movement by single kinesin molecules when intermediate states in the hydrolysis cycle are prolonged with ATP analogues or inhibitors. In contrast to myosin and dynein, kinesin with bound ADP dissociates from microtubules during translocation, whereas kinesin with unhydrolysed nucleotide remains tightly associated with the polymer. These findings imply that kinesin converts ATP energy into mechanical work by a pathway distinct from that of myosin or dynein.

Topics: Adenosine Diphosphate; Adenosine Triphosphatases; Adenosine Triphosphate; Aluminum; Aluminum Compounds; Animals; Cytoskeleton; Decapodiformes; Fluorides; Kinesins; Kinetics; Microtubules; Myosins; Vanadates