Na؉ /K ؉ -transport through mammalian cell membranes by Na ؉ /K ؉ -ATPase (EC 3.6.1.37) needs the interaction of ATP sites with different binding affinities during catalysis: one with catalytic (high affinity site) and one with regulatory properties (low affinity site). To find affinity labels for the latter one, the effects of 2-Odansylated ATP analogs on Na -ATPase binds ATP with high affinity to the sodium exporting E 1 -form (E 1 ATP binding site) and is consequently phosphorylated. After the release of sodium at the outer cell side and the following dephosphorylation, the enzyme needs a second binding of ATP. Therefore, ATP binds with low affinity to the E 2 -form (E 2 ATP binding site) of Na ϩ /K ϩ -ATPase and enhances the rate-limiting step of deocclusion of potassium during import (2, 3). In contrast to expectations deriving from this single ATP site model with its subsequent formation of the ATP sites, use of substitution-inert MgATP complex analogs has led to the postulate of a coexistence (in time and at different places) of both ATP sites (4). The Repke-Schön-Stein model (5) attempts to explain such a situation by shifting the energy excess of the sodium-transporting subunit to the potassium-transporting subunit. Each subunit follows a whole Albers-Post cycle but 180°out of phase. The bicyclic model of Plesner, on the other hand, gets its power from two ATP binding sites whose partial activities (Na ϩ -ATPase, K ϩ -phosphatase) are lower than the overall reaction (Na ϩ /K ϩ -ATPase). A single subunit does not have to pass all of the intermediates of the Albers-Post circle, but the sum fulfills all steps required for a whole turnover (6). However, there is still a lot of discussion about the intermediates shared by the partial reactions and the overall reaction (7,8).Substitution-inert MgATP complex analogs like CrATP 1 or CoATP are helpful tools to dissect the overall Na ϩ /K ϩ -ATPase activity by specific modifications of either the E 1 ATP site or the E 2 ATP site (9, 10). The activities of the E 1 ATP binding site (for example ATP/ADP exchange and "frontdoor phosphorylation") are unaffected by the inactivation of the E 2 ATP binding site by Co(NH 3 ) 4 PO 4 (11). Similarly, CrAMP-PCP, which inactivates the E 1 ATP site but is unable to phosphorylate it, does not affect activities of the E 2 ATP site, namely 86 Rb ϩ occlusion, K ϩ -activated phosphatase activity, and "backdoor phosphorylation" (12, 13). Although substitution-inert metal ATP complexes are on the one hand helpful tools to get information on basic prop-

A direct approach to enzyme activity measurements is presented. Vibrational spectroscopy can monitor the progress of enzymatic reactions because the vibrational spectrum of substrates and products usually differs. This is demonstrated by the example of ATP hydrolysis by Ca(2+)-ATPase: The substrate concentration can be followed using the infrared absorption of the alpha- and beta-PO(2)(-) phosphate groups of ATP, and the product concentration can be followed using the PO(3)(2-) absorption of P(i) and of the beta-phosphate of ADP. The results of the infrared spectroscopic measurement of ATPase activity and of an independent activity assay agree very well. The main advantage of the infrared method is that it observes the reaction of interest directly--that is, no activity assay that converts the progress of the reaction into an observable quantity is required.

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A direct approach to enzyme activity measurements is presented. Vibrational spectroscopy can monitor the progress of enzymatic reactions because the vibrational spectrum of substrates and products usually differs. This is demonstrated by the example of ATP hydrolysis by Ca2+-ATPase: The substrate concentration can be followed using the infrared absorption of the α- and β-PO2− phosphate groups of ATP, and the product concentration can be followed using the P032- absorption of Pi and of the fl-phosphate of ADP. The results of the infrared spectroscopic measurement of ATPase activity and of an independent activity assay agree very well. The main advantage of the infrared method is that it observes the reaction of interest directly—that is, no activity assay that converts the progress of the reaction into an observable quantity is required.

Active Na ϩ /K ϩ -transport through mammalian cell membranes catalyzed by the sodium pump needs the interaction of high and low affinity ATP binding sites during catalysis (1). During pumping, a high affinity ATP site (E 1 ATP site) 1 is phosphorylated when Na ϩ /K ϩ -ATPase (EC 3.6.1.37) is in its Na ϩ -exporting E 1 conformational state. Dephosphorylation, however, turns the enzyme to the K ϩ -importing E 2 conformation that binds ATP with low affinity (E 2 ATP site) (for a review, see Ref. 2). The kinetics of substrate hydrolysis of the enzyme vary with the nature of the nucleoside triphosphate. Although ATP hydrolysis proceeds in a negative cooperative way (3), inhibition of ATP hydrolysis by 2Ј,3Ј-O(2,4,6-trinitrophenyl)-ATP (TNP-ATP), a substance that is not hydrolyzed, was reported to be partially competitive and noncompetitive (4). Moreover, 2Ј(3Ј)-O(6-NЈ,NЈ-dimethylaminonaphthalenesulfonyl)-ATP (DANS-ATP) and 8-N 3 -DANS-ATP, which are not hydrolyzed either, show a positive cooperative effect during interaction with Na ϩ /K ϩ -ATPase (1). MgATP complex analogs can discriminate between E 1 ATP and E 2 ATP binding sites (5). Although Cr(H 2 0) 4 ATP (Cr-ATP) inactivates the E 1 ATP binding site, Co(NH 3 ) 4 ATP (Co-ATP) inactivates the E 2 ATP site (6 -8). The ribosyl-modified TNP-ATP is known as a substance that binds in relation to ATP with increased affinities to both ATP binding sites (4, 9). Furthermore, we showed recently that ribosyl-modified DANS-ATP binds with much higher affinity to the E 2 ATP site than to the E 1 ATP site (1). This peculiar phenomenon is not understood very well. A better understanding would be helpful not only to find more protein-reactive ATP derivatives with a preference for the low affinity E 2 ATP binding site but also to realize whether the method of analysis of the complex kinetics with a Koshland-Némethy-Filmer model of two cooperating ATP sites is generally applicable to all ATP derivatives. Hence, such a model would describe a general property of the enzyme. This would also include that it is justified to extrapolate from the knowledge of microscopic dissociation constants of the E 1 ATP and E 2 ATP sites, obtained from the inactivation with MgATP complex analogs (5), to the complex macroscopic kinetics of Na ϩ /K ϩ -ATPase (1). Therefore, we started a careful kinetic analysis of a number of ATP and nucleoside analogs with modified ribose and polyphosphate moieties. Analysis of all of the substances for their microscopic dissociation constants of the E 1 ATP and E 2 ATP sites by previously reported methods (1, 5) and of the kinetics of overall hydrolysis or substrate inhibition by use of a model of two interacting ATP sites revealed that the previously published Koshland-Némethy-Filmer model describes sufficiently well all kinetics. The correlation of kinetic data with structural data led to a postulate of minimal requirements of * This work was supported by the Deutsche Forschungsgemeinschaft through the Graduiertenkolleg "Molekulare Biologie und Pharmakologie" Gies...