The stereochemical course of phosphoric residue transfer during the myosin ATPase reaction.

When adenosine 5'-(3-thiotriphosphate), stereospecifically labeled in the gamma position with 18O, was hydrolyzed in the presence of myosin subfragment 1 in 17O-enriched water, the product inorganic [16O,17O,18O]thiophosphate was chiral. The configuration of this product showed that the hydrolysis proceeds with inversion at the transferred phosphoric residue. This result suggests a direct, in-line hydrolysis mechanism for the ATPase.

The hydrolysis of MgATP catalyzed by myosin and its fragments obtained by proteolysis occurs via reversible cleavage of ATP followed by rate-limiting release of products. This is supported by quenched flow studies described in Trentham et al. (1976) by the rapid exchange of water oxygens into the y-phosphoric residue of ATP and Pi (Levy and Koshland, 1959;Bagshaw et al., 1975) and by positional isotope exchange studies (Geeves et al., 1980). No evidence for a phosphoenzyme intermediate has been obtained from oxygen isotope studies (Sartorelli et al., 1966;Trentham, 1977). However, evidence for the involvement of a phosphoenzyme was presented by Kinoshita et al. (1969) but disputed by Wolcott and Boyer (1973).
The steady state intermediates, protein-bound ATP and ADP. P, interconvert rapidly (relative to product release) in the active site, but the mechanism of this chemical step has not been determined. Important information to elucidate this chemical mechanism is the stereochemical course of phosphoric residue transfer from nucleoside triphosphate to water. Put most simply: does this transfer occur with retention or inversion of configuration at the y-phosphorus? The techniques to study the stereochemistry of phosphoric residue transfer to species other than water have been developed recently and reviewed by Knowles (1980). In these cases, the oxygens of the transferred phosphoric residue are labeled differently, using three different oxygen isotopes (-P1eOL70180) or by using sulfur as an analog of oxygen (-PSL"O1*O). The results of the two methods have been compared in the case of glycerol kinase and were in agreement (Pliura et al., 1980). The accumulated data on phosphoric residue transfer suggest strongly that, when the residue is transferred directly between * This work was supported by Grant AM 23030 from the National Institutes of Health, and grants from the Muscular Dystrophy Association of America and the Whitehall Foundation. ."P NMR spectra were obtained at the Middle Atlantic NMR Facility, which is supported by National Institutes of Health Grant RR 542 at the University of Pennsylvania. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ''advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Postdoctoral fellow of the Muscular Dystrophy Association of America.
substrates, the configuration is inverted. Retention of configuration occurs when there is a two-step transfer (usually via a phosphoenzyme), with presumably each step occurring with inversion. We have developed a method of determining the stereochemistry of phosphoric residue transfer to water (Webb and Trentham, 1980) by configurational analysis of chiral inorganic [1s0,170,180]thiophosphate. This paper describes the stereospecific hydrolysis of ~y-180;y-1sOl]ATPyS1 in [170]water in the presence of myosin and analysis of the product inorganic [160,170,'sO]thiophosphate. This allows us to answer the problem formulated in Equation 1' as to whether the reaction proceeds with inversion or retention of configuration (or possibly racemization).

(retention) (inversion)
The analysis depends on there being little or no oxygen exchange between the product and water during the ATPyS hydrolysis. Whether or not oxygen exchange occurs in this reaction was not known and so is investigated.
&-1BO;y-'801]ATPyS was prepared by the method of Richard and Frey (1978), outlined in Scheme 1. [''OO,]AMP(S) was prepared by a modification of the method of Murray and Atkinson (1968). Thiophosphoryl chloride (4 mmol) was added to adenosine (1 mmol) and dissolved in triethylphosphate (10 ml) at 0°C. The mixture was left at 0°C overnight. Excess thiophosphoryl chloride was then removed by rotary evaporation and ["O]water (1 m l ) was added. The solution was left at room temperature for 1 h before dilution with unlabeled water. The [180~]AMP(S) was purified by ion exchange chromatography on a column of DEAE-cellulose (40 X 3 cm in diameter), with elution by a gradient (1.21) of triethylammonium bicarbonate from 10 to 400 The abbreviation used is: ATPyS, adenosine 5"(3-thiotriphosphate). Other thionucleotides are abbreviated similarly. The   (Sheu and Frey, 1977;Jaffe and Cohn, 1978). It follows that the y-"0 in the product ATPyS is stereospecifically attached. (Richard and Frey, 1978). The '#O enrichment of the AMP(S) was 75% in the two labeled positions as determined by "P NMR using the uptield shift in "'P resonance due to "0 (Cohn and Hu, 1978;Lowe and Sproat, 1978 Routine 31P NMR spectra were recorded at 24.3 MHz on a Varian NV 14 spectrometer modified to operate in the Fourier transform mode and equipped with a multinuclear probe and with quadrature phase detection. 31P NMR spectra to determine I8O content and distribution were recorded at 145.7 MHz on a Bruker WH 360/180 spectrometer, equipped with a dueterium field lock and operating in the Fourier sample tube (Wilmad Glass Co.) and was maintained at 22°C. Spectral transform mode. The sample solution (1.5 ml) was in a 1-cm diameter width was lo00 Hz, with a pulse width of 15 ps and an acquisition time of 8.2 s. A sensitivity enhancement exponential function gave a line broadening of 0.1 Hz.

RESULTS AND DISCUSSION
To determine whether any product water-oxygen exchange occurred during ATP@ hydrolysis in the presence of subfragment 1, unlabeled ATPyS was hydrolyzed in "0-enriched water. A 31P N M R spectrum of the product inorganic thiophosphate (with a signal to noise ratio of 20) showed that only 1 "0 atom per molecule had been incorporated into the product. Thus, no exchange occurs. This is probably because the cleavage of ATPyS is the rate-limiting step of the catalytic me~hanism.~ In [170]water, the hydrolysis of the ATPyS, stereospecifically labeled with "0 in the y-position will give rise to inorganic  Fig. la. The assignment of the peaks is described by Webb and Trentham (1980). The spectrum of the ATPpS derived from the myosin hydrolysis product in Fig. lb shows that there is an excess of Previously, it had been shown that protein-bound ATP@ is the major steady state intermediate (Bagahaw et al., 1972). This evidence, together with the lack of oxygen exchange, is best explained by the cleavage of ATPyS being rate-limiting. This is in contrast to the hydrolysis of ATP. In the latter case, the cleavage is some 2 orders of magnitude faster and product release is rate-limiting. This allows the ATP and ADP.P, to interconvert many times before the products are released. A marked decrease in overall catalytic rate for ATPyS relative to ATP has been observed in many enzymic systems (Eckstein, 1975). species 1 over 2. Hence, there was an excess of the R enantiomer of inorganic thiophosphate, which means that myosin subfragment 1 catalyzed the hydrolysis of ATPyS with inversion (Equation 1). The calculation below indicates that nearly all of species 2 arises because of the lack of isotopic purity.
From the estimated isotopic enrichments (75% for IMO in ATPyS and 45% for I7O in water, which also contains 12% The small difference between the calculated and observed ratios may be due to incomplete stereospecificity of the myosin ATPase reaction. If the product inorganic thiophosphate contained the R and S enantiomers in a ratio of 18:1, the ratio of ATPpS species would have been 38:35:23:4. However, a more likely explanation for the difference is that, during the incorporation of inorganic thiophosphate into ATPPS, there was partial hydrolysis of the intermediate, glycerate I-thiophosphate 3-phosphate. This yields inorganic thiophosphate, which on reincorporation results in loss of isotope and a redistribution in the relative populations of ATPpS species. We have observed loss of isotope at this stage previously. Twelve per cent hydrolysis would cause the peak ratio to be 40:34:22:4. To minimize this problem, it is crucial that the reaction catalyzed by glyceraldehyde phosphate dehydrogenase in the incorporation procedure (Webb and Trentham, 1980) is rate-limiting. the R enantiomer of inorganic ['60,'70,'80]thiophosphate means that the hydrolysis proceeds with inversion of configuration. It is probable that the chemical mechanisms of ATP and ATPyS hydrolysis are similar, so that the most likely mechanism for ATP hydrolysis is direct, in-line displacement of ADP by a water oxygen. The results described here, together with the evidence cited above, point toward phosphoric residue transfer during ATP hydrolysis being a single characterizable step that is reversible and rapid, relative to the overall myosin ATPase catalytic center activity.
This result is strong evidence against a phosphoenzyme being an intermediate on the reaction pathway. Tsai and Chang (1980) have shown that nucleotidase catalyzes the hydrolysis of AMP@), also with inversion of configuration. It will be of great interest to see whether, in the sarcoplasmic reticulum Ca-ATPase for which there is strong evidence for a phosphoenzyme intermediate (Yamamoto and Tonomura, 1968), the hydrolysis of ATPyS proceeds with retention of configuration. The question of a phosphoenzyme intermediate in the mitochondrial ATPase remains open. The application of this stereochemical approach is likely to provide evidence for or against phosphoenzyme intermediates in nucleoside triphosphate-requiring, energy-transducing systems.