Proton inventory technique

It must be appreciated that the selection of the best model—that is, the best equation having the form of Eq. (6-97)—may be a difficult problem, because the number of parameters is a priori unknown, and different models may yield comparable curve fits. A combination of statistical testing and chemical knowledge must be used, and it may be that the proton inventory technique is most valuable as an independent source capable of strengthening a mechanistic argument built on other grounds. 

At its best, the study of solvent kies by the formalism given can be used to learn about proton content and activation in the transition state. For this reason it is known as the proton inventory technique. The kinetics of decay of the lowest-energy electronic excited state of 7-azaindole illustrates the technique.25 Laser flash photolysis techniques (Section 11.6) were used to evaluate the rate constant for this very fast reaction. From the results it was suggested that, in alcohol, a double-proton tautomerism was mediated by a single molecule of solvent such that only two protons are involved in the transition state. In water, on the other hand, the excited state tautomerism is frustrated such that two water molecules may play separate roles. Diagrams for possible transition states that can be suggested from the data are shown, where of course any of the H s might be D s. 

The proton inventory technique was applied to this system in an attempt to verify further these suggestions. The rate constants for the reactions were evaluated in CH3OH/CH3OD, C2H5OH/C2H5OD, and H2O/D2O mixtures over the full range of deuterium mole fractions. The data for methanol and water are given in Table 9-8. 

Note that prior applications of the proton inventory technique were to reactants in their ground states. The particular example cited, however, refers to an excited state molecule (and indeed was the first of its kind). An implicit assumption made in the... 

Proton inventory technique. 21.9-220 Pseudo-first-order kinetics, 16 Pulse-accelerated-flow method. 255 Pulse radiolysis, 266-268 Pump-probe technique. 266... 

PROTON INVENTORY TECHNIQUE Dolichyl-phosphate mannosyltransferase,... 

Proton flow to ATP synthesis/hydrolysis, BINDING CHANGE MECHANISM PROTON INVENTORY TECHNIQUE... 

PROTON INVENTORY TECHNIQUE Transition-state mimic,... 

In proton inventory technique, solvent isotope effects are plotted against atomic fractions of deuterium in mixed isotopes of water. A linear plot represents a contribution from a single origin, whereas nonlinear plots may be generated from multiple origins. 

Propagation of errors, 40, 48, 248 Propinquity effect, 263, 365 Protol5Tsis, 147, 148 Proton inventory technique, 302 Proton transfer, 166 direct, 148 extent of, 346 fast, 97, 146, 173 isotope effect in, 296 partial, 395 Proximity effect, 365 Pseudo-first-order rate constant, 23 Pseudo-first-order reaction, 61 Pseudo-order rate constant, 23 Pseudo-order reaction, 23 Pseudo-order technique, 26, 78 Pulse NMR, 170... 

Solvent kinetic isotope effects (SKIEs) in H2O/D2O mixtures on the reaction of /)NPP catalyzed by calcineurin gave a small normal value of 1.35. Proton inventory and fractionation data are consistent with a mechanism involving a single proton transfer from a metal-bound water, although due to the small KSIE value and the inherent experimental error of the proton inventory technique, the participation of a second proton could not he excluded. Further information has been furnished by heavy-atom isotope effects. Reaction of NPP catalyzed by APP shows that phosphoryl transfer is fully rate limiting. However, for calcineurin the... 

Interpreting solvent kinetic isotope effects in terms of these interactions can provide information about the transition structure, its solvation, or both. With one technique, known as the proton inventory technique, the rate of a reaction is studied in a series of mixtures of H2O and D2O. The isotope effect, / H20/ is plotted versus the parameter n, the mole fraction of deuterium in the solvent. The slope and curvature of the resulting plot are then analyzed for information about the number and role of solvent molecules involved in the rate-limiting step. ° ... 

NMR spectroscopy is one among many probes that have been reported for the evaluation of kinetic isotope effects. Several NMR methods, as analogs of previous proton inventory techniques involving classical kinetic methods were reported, involving line-shap>e analyses and polarization transfer experiments on the exchanging protons or deuterons and/ or on the remote spins as functions of the deuterium atom fraction n in the mobile proton sites. Moreover, the kinetic isotope effects and the number of transferred protons originating from... 

Fig. 11.7 Proton inventory curves (plots of k(n)/k(H) vs. n (or x) = atom fraction D) for overall isotope effects of 2 (upper plot) and 10 (lower plot). In each, the three curves (reading from top to bottom) are for single site, two site, and multi-site isotope effects. Error bars of 3% middle curves) are shown in each case. For ko/ki = 2 the technique is unable to distinguish between the curves at this level of precision (3%), but is more than adequate for ko/ki = 10 (Schowen, R. L., J. Label Compd Radiopharm. 50, 1052 (2007), with permission Wiley Interscience)...

This technique is called the proton inventory method and has been employed with success in studies of enzyme reactions. Numerous examples are cited by Quinn (reading list). 

By proton inventory, a technique that determines whether acid and base groups act simultaneously, we found that hydrolysis of 36 by artificial enzyme 44 involves two protons moving in the transition state [130]. Thus, ImH+ of 46 is hydrogen bonded to a phosphate oxyanion of bound substrate 36 water hydrogen bonded to the Im then attacks the phosphorus, and as the O-P bond forms the ImH+ proton transfers (along with the water proton) to produce the phosphorane monoanion 47. This then goes on to the cleaved product in later catalyzed steps before there is time for pseudo-rotation. These general conclusions have been described and summarized in several publications [131-137]. 

Although at pH 8 the electron distribution favours the formation of flavin semiquinone and reduced iron-sulfur center, the magnetic moments of the two redox centers do not interact. At pH 10, however, 2-electron-reduced TMADH exhibits the EPR spectrum diagnostic of the spin-mteracting state. In a more detailed analysis using the pH-jump technique, the interconversion of three states of TMADH [state 1, dihy-droflavin-oxidised 4Fe-4S center (formed at pH 6) state 2, flavin semi-quinone-reduced 4Fe-4S center (formed at pH 8) state 3, spin interacting state (formed at pH 10)] were studied in both H2O and D2O (Rohlfs et al., 1995). The kinetics were found to be consistent with a reaction mechanism that involves sequential protonation/deprotonation and electron transfer events (Figure 6). Normal solvent kinetic isotope effects were observed and proton inventory analysis revealed that at least one proton is involved in the reaction between pH 6 and 8 and at least two protons are involved between pH 8 and 10. At least three protonation/... 

The inventory technique is clearly a potentially useful tool for elucidating enzymatic transition states. Deacetylation of acetyl-a-chymotrypsin is consistent with a single proton transfer (Eqn. 45) [25] with a fractionation factor 0.42. It is thought that this result excludes the charge-relay mechanism (Eqn. 46) which would require a quadratic term for the dependence. 

We had invoked a simultaneous two proton transfer mechanism rather than a sequential mechanism - in which one catalytic group followed the other in the overall process - and were able to test this with a technique called proton inventory." We examined the original cyclodextrin fcfv-imidazole in mixtures of water and D2O and saw that the rate constant as a function of deuterium concentration followed a curved line, indicating that the isotope effect involved two different protons rather than a single one. To validate this, we also examined the same kind of plot with the cyclodextrin mono-imidazole, in which only one proton would be expected to be moving in the transition state, and this indeed followed a linear plot supporting a single proton motion in the isotope effect. 

The pH vs rate profile showed a bell-shaped curve indicating that this catalyst uses both B and BH+ in a bifunctional mechanism. As with the enzyme, the bis-imidazole catalyst can perform its bifunctional catalysis by a simultaneous mechanism, not the sequential mechanism of simple buffer catalysis. We saw that this was indeed the case, as revealed by the tool called "proton inventory." In this technique the reaction is performed in D2O, in H2O, and in mixtures of the two. If only one proton that can exchange with D2O is moving in the transition state, the points all lie on a straight line between the H2O and slower D2O points. If two (or more) protons are moving, the line is curved. It had been found for the enzyme ribonuclease A [10] that a curved line was seen corresponding to the movement of two protons, and we also saw a curved plot—with very similar data— for our cyclodextrin-6A,6B-bisimidazole catalyst 6 [11]. Controls established that indeed this was a reliable indication that our system is performing simultaneous bifunctional catalysis, just as the enzyme does. In particular, the... 

Many reaction mechanisms involve the movement of one or more protons, so this technique is applicable to a vast number of reactions, particularly so in enzymology, where catalysis often involves the coupling of several proton transfers in a single step (see Chapter 9). Therefore, the proton inventory is an extremely powerful physical organic chemistry technique that has found use in an untraditional physical organic field. 

Work on the enzyme had included the study of its proton inventory, in which rates are determined in H2O and D2O mixtures. The results indicated that two protons move in the rate determining step, consistent with a simultaneous bifimctional mechanism. It was therefore gratifying that in our synthetic model enzyme the same technique also showed a two-proton simultaneous mechanism, as in the enzyme. The general lesson is that it is... 

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