A probe has been developed that can rapidly measure micromolar concentrations of inorganic phosphate (Pi), in particular to follow the release of Pi in real time from enzymes such as phosphatases. Its application is described to investigate the mechanism of actomyosin subfragment 1 ATPase. The probe uses the A197C mutant of Escherichia coli phosphate binding protein (PBP), generated by oligonucleotide-directed mutagenesis. A new fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC), was attached to the single cysteine to produce the reporter molecule that was purified free of unlabeled protein and unattached MDCC. The labeled protein has an excitation maximum at 425 nm and emission maximum at 474 nm in the absence of Pi, shifting to 464 nm with a 5.2-fold increase in fluorescence (lambda max/lambda max) when complexed with Pi at pH 7.0, low ionic strength, 22 degrees C. The fluorescence increase is not much altered by change to pH 8 or by increase in ionic strength to 1 M. Pi binds tightly (Kd approximately 0.1 microM) and rapidly (1.36 x 10(8) M-1 s-1) and the dissociation rate constant is 21 s-1, at pH 7.0, low ionic strength, 22 degrees C. A variety of phosphate esters were tested to investigate the specificity of the MDCC-PBP and none gave a significant fluorescence increase at 100 microM or higher concentration. ATP weakly inhibited the Pi-induced fluorescence change, indicating that it binds at least 3000-fold weaker than Pi. Because Pi is a widespread contaminant, the probe is used in conjunction with a "Pi mop", consisting of 7-methylguanosine and purine nucleoside phosphorylase, to remove free Pi from solutions by its conversion to ribose 1-phosphate. Because the equilibrium constant of this reaction is > 100, free Pi can be reduced below 0.1 microM. The probe was used to measure the rate of Pi release during a single turnover of ATP hydrolysis with actomyosin subfragment 1 from rabbit skeletal muscle, to determine to what extent Pi release contributes to the rate limitation of this ATPase. Using a stopped-flow apparatus, a small lag prior to rapid Pi release was detected at pH 7.0, low ionic strength, between 5 and 22 degrees C at both high and low [ATP]. For measurements of a single turnover at low [ATP], the observed rate increased with [actin], showing saturation with a Km with respect to actin of 26 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Direct measurement of the kinetics of kinesin dissociation from microtubules, the release of phosphate and ADP from kinesin, and rebinding of kinesin to the microtubule have defined the mechanism for the kinesin ATPase cycle. The processivity of ATP hydrolysis is ten molecules per site at low salt concentration but is reduced to one ATP per site at higher salt concentration. Kinesin dissociates from the microtubule after ATP hydrolysis. This step is rate-limiting. The subsequent rebinding of kinesin · ADP to the microtubule is fast, so kinesin spends only a small fraction of its duty cycle in the dissociated state. These results provide an explanation for the motility differences between skeletal myosin and kinesin.Kinesin, a microtubule-activated ATPase, functions as a cytoplasmic motor to drive organelle translocation toward the plus ends of microtubules 1-3 . The principles governing the conversion of chemical energy from ATP hydrolysis to force production for the sliding of kinesin along microtubules may be similar to those for actomyosin and the axonemal dynein-microtubule ATPases 4,5 . However, the motility of single motor molecules in vitro suggests that mechanochemical coupling for kinesin must be somewhat different from actomyosin. For example, a single molecule of kinesin (2 heads) will promote translocation for several micrometres and at maximal rates 6 . In contrast, multiple skeletal myosin molecules are required for directed movement along an actin filament and the velocity increases as the number of myosin molecules is increased 7 . In addition, the non-hydrolysable ATP analogue AMP-PMP (β, γ-imidoadenosine 5′-triphosphate) causes dissociation of the actomyosin and microtubule-dynein complexes, but promotes stabilization of the microtubule-kinesin complex 5,8 .Here we describe mechanistic studies of the kinetics of indi|vidual steps in the ATPase cycle of the kinesin ATPase to explain the interactions of kinesin with the microtubule lattice responsible for movement. We have used the Drosophila kinesin motor domain expressed in Escherichia coli 9-11 . This protein, designated K401 and containing the N-terminal 401 amino acids, is a fully active, homogeneous preparation with the kinetic and structural properties expected of a native kinesin 9-11 . It has a very low ATPase activity in the absence of microtubules which is limited by the rate of ADP release (∼0.01 s −1 ). In the presence of microtubules, the steady-state rate increases to a maximum of 20 ± 2 s −1 . Furthermore, K401 is a dimer under our experimental conditions (J. J. Correia, S.P.G., M. L. Moyer and K.A.J., manuscript submitted).Chemical quench-flow experiments 11 established the rate of ATP hydrolysis at the active site to be significantly faster (100 s −1 ) than steady-state turnover; therefore, the rate-limiting step † Present address: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA. § To whom correspondence should be addressed. of the microtubule-activated ATPase must occur after ATP h...

The mechanism of Pi interaction with phosphate binding protein of Escherichia coli has been investigated using the A197C mutant protein labeled with a coumarin fluorophore (MDCC-PBP), which gives a fluorescence change on binding Pi. A pure preparation of MDCC-PBP was obtained, in which the only significant inhomogeneity is the presence of equal amounts of two diastereoisomers due to the chiral center formed on reaction of the cysteine with the maleimide. These diastereoisomers could not be separated, but Pi binding data suggest that they differ in affinity and fluorescence change. When Pi binds to MDCC-PBP, the fluorescence quantum yield increases 8-fold and the fluorescence intensity at 465 nm increases 13-fold. The kinetics of Pi binding show saturation of the rate at high Pi concentrations, and this together with other information suggests a two-step mechanism with the fluorescence change after binding, concomitant with a conformational change of the protein that closes the cleft containing the Pi binding site. Cleft closure has a rate constant of 317 s-1 (pH 7.0, 5 degrees C), and opening has a rate constant of 4.5 s-1. The fluorescence increase is likely to arise from a change in the hydrophobic environment during this closure as the steady state fluorescence emission (lambdamax and intensity) on Pi binding is mimicked by the addition of ethanol to aqueous solutions of an MDCC-thiol adduct. Fluorescence lifetimes in the absence and presence of Pi were 0.3 and 2.4 ns, respectively, consistent with the change in quantum yield. The rotational correlation time of the coumarin increases only 2-fold from 15 to 26 ns on binding Pi as measured by time-resolved polarization, consistent with the main rotation being determined by the protein even in the open conformation, but with greater local motion. Circular dichroism of the coumarin induced by the protein is weak in the absence of Pi and increases strongly upon saturation by Pi. These data are also consistent with an open to closed conformational model.

1. The rate of appearance of inorganic phosphate (Pé) and hence the ATPase activity of rabbit psoas muscle in single permeabilized muscle fibres initially in rigor was measured following laser flash photolysis of the P 3 -1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP) in the presence and absence of Ca¥. Pé appearance was monitored from the fluorescence signal of a Pé-sensitive probe, MDCC-PBP, a coumarin-labelled A197C mutant of the phosphate-binding protein from Escherichia coli. Fibres were immersed in oil to optimize the fluorescence signal and to obviate diffusion problems. The ATPase activity was also measured under similar conditions from the rate of NADH disappearance using an NADH-linked coupled enzyme assay. 2. On photolysis of NPE-caged ATP in the presence of Ca¥ at 20°C, the fluorescence increase of MDCC-PBP was non-linear with time. ATPase activity was 41 s¢ in the first turnover based on a myosin subfragment 1 concentration of 150 ìÒ. This was calculated from a linear regression of the fluorescence signal reporting 20-150 ìÒ of Pé release. Tension was at 67% of its isometric level by the time 150 ìÒ Pé was released. ATPase activities were 36 and 31 s¢ for Pé released in the ranges of 150-300 ìÒ and 300-450 ìÒ, respectively. The ATPase activity had a QÔÑ value of 2·9 based on measurements at 5, 12 and 20°C. 3. An NADH-linked assay showed the ATPase activity had a lower limit of 12·7 s¢ at 20°C.The response to photolytic release of ADP showed that the rate of NADH disappearance was partially limited by the flux through the coupled reactions. Simulations indicated that the linked assay data were consistent with an initial ATPase activity of 40 s¢. 4. On photolysis of NPE-caged ATP in the absence of Ca¥ the ATPase activity was 0·11 s¢ at 20°C with no discernible rapid transient phase of Pé release during the first turnover of the ATPase. 5. To avoid the rigor state, the ATPase rate in the presence of Ca¥ was also measured on activation from the relaxed state by photolytic release of Ca¥ from a caged Ca¥ compound, nitrophenyl-EGTA. At 5°C the ATPase rate was 5·8 and 4·0 s¢ in the first and second turnovers, respectively. These rates are comparable to those when NPE-caged ATP was used. 6. The influence of ADP and Pé on the ATPase activities was measured using the MDCC-PBP and NADH-linked assays, respectively. ADP (0·5 mÒ) decreased the initial ATPase rate by 23%. Pé (10 mÒ) had no significant effect. Inhibition by ADP, formed during ATP hydrolysis, contributed to the decrease of ATPase activity with time. 7. The MDCC-PBP assay and NPE-caged ATP were used to measure the ATPase rate in single permeabilized muscle fibres of the semitendinosus muscle of the frog. At 5°C in the presence of Ca¥ the ATPase activity was biphasic being 15·0 s¢ during the first turnover (based on 180 ìÒ myosin subfragment 1). Tension was 74% of its isometric level by the time 180 ìÒ Pé was released. During the third turnover the ATPase rate decreased to about 20% of that during the first turnover. 8. ATPase activity in isometr...