Kinetic competence

In tlie previous section we showed tliat because tlie stmcture space is very sparse tliere have to be many sequences tliat map onto tlie countable number of basins in tlie stmcture space. The kinetics here shows tliat not all tlie sequences, even for highly designable stmctures, are kinetically competent. Consequently, the biological requirements of stability and speed of folding severely restrict tlie number of evolved sequences for a given fold. This very important result is schematically shown in figure C2.5.4. 

The induced oxidation of iodide reveals that some intermediate is more kinetically competent toward iodide oxidation than either HCr04 or VOJ. The stoichiometric coefficients in Eq. (5-2) suggest that chromium(V) is the species responsible. 

These findings imply that, in the case of secondary radicals, the trapping with a second equivalent of Cp2TiCl can compete with tetrahydrofuran formation, and that -hydride elimination can kinetically compete with protonation of Ti - C bonds under our protic conditions [65,66,73,74], It remains to be seen how the reaction can be completely driven toward the tricyclic system 60 by ligand variation of the catalyst. 

Markovic and Hartwig isolated and characterized the first intermediate in iridium-catalyzed allylic substitution [100]. They isolated the metalacyclic iridium-phosphor-amidite fragment containing COD and the olefinic portion ofN- l -phenylallyl)aniline, the product of the allylic substitution reaction between cinnamyl carbonate and aniline (5 in Scheme 22). This complex containing the product of allylic substitution was first detected by NMR spectroscopy during catalytic reactions. It was then isolated, prepared independently, and shown to be chemically and kinetically competent to be an intermediate in allylic substitutions. 

Additional mechanistic insights were gained when Hartwig and coworkers isolated and characterized the first 7t-allyl complexes that are chemically and kinetically competent to be intermediates in iridium-catalyzed allylic substitution [46]. These complexes were prepared independently from allylic electrophiles that are more reactive than allylic carbonates. The isolation and structural characterization of these species provided a detailed view into the origins of enantioselectivity. 

Kt = 0.84 and at pH 7, KT = 0.36. The rate of ring-chain interconversions obtained from temperature-jump kinetic experiments appears to be hydro-nium-ion-catalyzed in the pH range 11.5-13, with rate constants reaching about 105 s 1. It was presumed that the cyclic tautomers 54 can serve as kinetically competent intermediates for transimination sequences at the active sites of pyridoxal-P-dependent enzymes. 

To account for the reactivity of 19 toward oxygen atom donors, a catalytic cycle was proposed. In this mechanism, the diferrous complex reacts with an oxygen donor to give an adduct with 19 that can either act as the atom transfer species or collapse to a ferry 1 species [Fe4+ = 0] intermediate. The data currently available does not allow for a more detailed description, although spectroscopic characterization and determination of the kinetic competence of the observed intermediate will allow the differentiation of several possible pathways. 

Identification of radical 3 as a species that is present in the steady-state phase of the reaction does not prove that it is an intermediate—it could be a species that is peripheral to the real reaction mechanism. Proof that a species is an intermediate requires a demonstration that it is kinetically competent to participate in the mechanism. In the case of a metastable radical, the usual procedure is to conduct transient kinetic studies using a rapid mixing apparatus equipped to quench samples by spraying them into liquid isopentane. The frozen aqueous samples (snows) from the timed cold quenches are then packed into EPR tubes and analyzed spectroscopically. Simple mixing of enzyme with SAM and lysine followed by freeze-quenching on the millisecond time scale does not work because the activation by SAM takes about 5 s. However, a preliminary mix of enzyme with SAM and [2- C]lysine, aging of the solution for 5 s within the apparatus. 

The finding of kinetically competent radical intermediates in the reaction catalyzed by LAM mandates an efficient mechanism to generate them. The results of tritium transfer experiments in conjunction with the similarity between the reaction catalyzed by LAM and... 

B12-dependent ribonucleotide reductase was the first enzyme for which the rate of AdoCbl homolysis was examined. Pioneering studies by Blakely and co-workers established that a homolytic mechanism was indeed operating, as opposed to heterolytic cleavage to give Co(I), and that the rate of homolysis (k= 38s" ) was kinetically competent (Tamao and Blakely, 1973). Subsequently, the kinetic competency of CooC cleavage in ethanolamine deaminase was established (Hollaway et al., 1978), and, as discussed below, homolysis has been found to be far more rapid than turnover for all the enzymes so far examined. 

Gerfen, G. J., Licht, S., Willems, J. P., Hoffman, B. M., and Stubbe, J., 1996, Electron paramagnetic resonance investigations of a kinetically competent intermediate formed in ribonucleotide reduction Evidence for a thiyl radical-Cob(II) alamin interaction. J. Am. Chem. Soc. 118 819298197. 

This red species has also been shown to form in the reaction of reduced enzyme with DMSO (Adams et at., 1999), and appears to represent a kinetically viable DMSO Michaelis complex, which rapidly achieves equilibrium with the eorresponding Eox DMS species in the enzyme active site. Consistent with this, recent kinetic work with DMSO reductase has demonstrated that the enzyme is kinetically competent to oxidized DMS as well as reduce DMSO (Adams et al., 1999). Interestingly, the former reaction is sensitive to extended preincubation of enzyme with DMS under aerobic (but not anaerobic), but the latter reaction is not. The spectrum of the DMS-modified enzyme resembles that exhibited by recombinant as-isolated enzyme heterologously expressed in E. coli, which can be converted to native enzyme by reduction and reoxidation (Hilton et al.,... 

For nonheme enzymes that fiuther activate dioxygen, it is apparent that the diferrous forms also bind O2 to eventually generate the active species responsible for the oxidative transformations. In the case of MMO, the first intermediate has been labeled compound P (Scheme 2), which subsequently converts to compound Q both are kinetically competent to hydroxylate methane. In the case of RNR, compound X (Scheme 1) is responsible for the one-electron oxidation of a tyrosine residue to generate a tyrosyl radical. Based on chemical considerations and its Mossbauer properties, it has been proposed that compound P is a diferric peroxide species. To date, three model complexes of compound P, with comparable spectroscopic properties, have been structurally characterized (Figure g). In two of these models O2 is bound in a cis... 

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