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)

A spectrophotometric method for the measurement of inorganic phosphate (Pi) has been developed by using 2-amino-6-mercapto-7-methylpurine ribonuleosde and purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltrnsferase, EC 2.4.2.1). This substrate gives an absorbance ase at 360 nm on phospborolyis at pH 6.5-8.5, and at pH 7.6 the change in extinction coefficient is 11,000 M-'-cmn'. The Michaelis-Menten constants of the two substrates with the enzyme are 70 pM for the nucleoside and 26 pAM for PI; the kept is 40 s-1 (25°C ments was purified further on Q-Sepharose. Actin and chymotryptic subfragment 1 were prepared from rabbit skeletal muscle as described (4, 5).MESG was synthesized by modification of the method of Broom and Milne (6). 2-Amino-6-chloro-purine ribonucleoside (2 g; Sigma) was dissolved in dry dimethylformamide (10 ml). Methyl iodide (4 ml) was added and the mixture was stirred for 14 h. Excess methyl iodide was removed in vacuo. Thiourea (2 g) was added and the mixture was stirred for 30 min. Methanolic ammonia was added until the solution became neutral (tested with pH indicator paper). The mixture was poured into stirred acetone to give a yellow precipitate. The solid was filtered and dried in vacuo. To ensure that the compound was pure with respect to other UV-absorbing compounds, it was dissolved in dimethylformamide and purified on a column of silica gel (2.5 x 15 cm), eluted with ethyl acetate/1-propanol/water (5:2:1; vol/vol). The compound was dried to a yellow solid by rotary evaporation and stored desiccated at -20TC. 1H NMR analysis was as expected (6).Samples of MESG were analyzed by HPLC using a C18 reverse-phase column (Whatman Partisil 10 ODS; 25 X 0.4 cm). Elution was at 2 ml-min-, using 10 mM potassium phosphate (pH 5.5) containing 10o (vol/vol) methanol. This system could also be used to monitor the base product of the phosphorylase reaction. The elution times are 7.5 min for MESG and 15 min for the base. RESULTSThe guanosine analog MESG has a strong UV absorbance peak at 330 nm at pH 7.6 (Fig. 1). In the presence of Pi and purine-nucleoside phosphorylase, the base 2-amino-6-mercapto-7-methylpurine forms, with the absorbance maximum shifted to 355 nm. Using the absorbance spectrum changes of MESG as a function of pH, it was shown that MESG has a pKa of 6.5 (Fig. 2). The base product has a pKa of 8.8 as determined by UV spectroscopy. This difference in ionization between base and nucleoside (Scheme II) is presumably due to the extra positive charge on the nucleoside. In the pH range 6.5-8.5, there is a UV absorbance difference between the phosphorylase substrate and product (Fig. 1).Abbreviation: MESG, 2-amino-6-mercapto-7-methylpurine ribonucleoside. 4884The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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...

Using a fluorescent sensor for inorganic phosphate, the kinetics of ATP hydrolysis by PcrA helicase were measured in the presence of saturating concentrations of oligonucleotides of various lengths. There is a rapid phase of inorganic phosphate release that is equivalent to several turnovers of the ATPase, followed by slower steady-state ATP hydrolysis. The magnitude of the rapid phase is governed by the length of single-stranded DNA, while the slow phase is independent of its length. A kinetic model is presented in which the rapid phase is associated with translocation along single-stranded DNA, after the PcrA binds randomly along the DNA. There is a linear relationship between the length of single-stranded DNA and both the duration and amplitude of the rapid phase. These data suggest that the translocation activity occurs at 50 bases/s in unidirectional single-base steps, each requiring the hydrolysis of 1 ATP molecule.

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.

We have measured the kinetics of inorganic phosphate (Pi) release during a single turnover of actomyosin nucleoside triphosphate (NTP) hydrolysis using a double-mixing stopped-flow spectrofluorometer, at very low ionic strength to increase the affinity of myosin-ATP and myosin-ADP-Pi to actin. Myosin subfragment 1 and a series of nucleoside triphosphates were mixed and incubated for approximately 1-10 s to allow NTP to bind to myosin and generate a steady state mixture of myosin-NTP and myosin-NDP-Pi. The steady state intermediates were then mixed with actin. The kinetics of Pi release were measured using a fluorescent probe for Pi, based on a phosphate binding protein [Brune et al. (1994) Biochemistry 33, 8262-8271]. These data are correlated with quenched-flow data, where the extent of the rapid burst of hydrolysis during the first turnover of ATP hydrolysis was followed by chemical quenching of the reaction mix at various times after rapidly mixing ATP and myosin subfragment 1. From the double-mixing actomyosin measurements, the kinetics of Pi release are biphasic. The fast phase corresponds to Pi release from the associated actomyosin-ADP-Pi complex. The slow phase measures the rate of the cleavage step on associated actomyosin. At saturating actin, there is a correlation between the amplitude of the fast phase and the size of the Pi burst observed by quenched flow in the absence of actin: the size of this phase corresponds to the amount of myosin-ADP-Pi formed during the first mix. For ATP at 20 degrees C the rate of the Pi release step is 75 (+/-5) s-1, 25-fold larger than the cleavage step, which is the rate-limiting step of actomyosin ATP hydrolysis at saturating actin. The rate constant of Pi release varies only slightly with nucleoside structure. The rate constant of the slow phase of the Pi release (measuring cleavage) is highly dependent upon the structure of the NTP substrate.

Superfamily 1B (SF1B) helicases translocate in a 5'-3' direction and are required for a range of cellular activities across all domains of life. However, structural analyses to date have focused on how SF1A helicases achieve 3'-5' movement along nucleic acids. We present crystal structures of the complex between the SF1B helicase RecD2 from Deinococcus radiodurans and ssDNA in the presence and absence of an ATP analog. These snapshots of the reaction pathway reveal a nucleotide binding-induced conformational change of the two motor domains that is broadly reminiscent of changes observed in other SF1 and SF2 helicases. Together with biochemical data, the structures point to a step size for translocation of one base per ATP hydrolyzed. Moreover, the structures also reveal a mechanism for nucleic acid translocation in the 5'-3' direction by SF1B helicases that is surprisingly different from that of 3'-5' translocation by SF1A enzymes, and explains the molecular basis of directionality.

The crystal structure of human rac1, a member of the rho family of small G-proteins, complexed with the non-hydrolysable GTP analogue, guanosine-5'-(beta gamma-imino)triphosphate (GMPPNP), has been determined by X-ray analysis at a resolution of 1.38 A. Comparison with the structure of H-ras indicates that rac1 has an extra alpha-helical domain that is characteristic of the rho G proteins, and may be involved in the signalling pathway of this family.