Pentacovalent transition state

Polynucleotide polymerases, or nucleotidyl transferases, are enzymes that catalyze the template-instructed polymerization of deoxyribo- or ribonu-cleoside triphosphates into polymeric nucleic acid - DNA or RNA. Depending on their substrate specificity, polymerases are classed as RNA- or DNA-dependent polymerases which copy their templates into RNA or DNA (all combinations of substrates are possible). Polymerization, or nucleotidyl transfer, involves formation of a phosphodiester bond that results from nucleophilic attack of the 3 -OH of primer-template on the a-phosphate group of the incoming nucleoside triphosphate. Although substantial diversity of sequence and function is observed for natural polymerases, there is evidence that many employ the same mechanism for DNA or RNA synthesis. On the basis of the crystal structures of polymerase replication complexes, a two-metal-ion mechanism of nucleotide addition was proposed [1] during this two divalent metal ions stabilize the structure and charge of the expected pentacovalent transition state (Figure B.16.1). 

The differing electronic configurations of silicon and carbon must also be taken into consideration. Silicon d orbitals are arranged in such a way as to make possible the formation of a pentacovalent or hexacovalent state. The hydrolytic cleavage of the Si—Cl bond, for example, may proceed through a pentacovalent transition state. The existence of silicon compounds with coordination numbers 5 and 6 can be substantiated through the same reasoning [3]. 

Now we will consider important information about the chirality of the reactant and the product that also distinguishes between the Sj j2 and S l mechanisms. The stereochemical consequences of the two mechanisms differ because the transition states in the two mechanisms differ. In the Sj,j2 mechanism, the nucleophile and the substrate form a pentacovalent transition state in the shape of a trigonal bipyramid. In the 1 mechanism, when the leaving group departs, the resulting carbocation is a planar, sp -hybridized carbocation. 

FIGURE 6.2 A two-metal catalytic mechanism proposed for the 3 — 5 -exonuclease reaction. Metal ion B (Mg ) stabilizes the pentacovalent transition state and the leaving 3 oxyanion. Metal ion A (Zn ) promotes the formation of hydroxide ion which is the attacking nucleophile. [After L. S. Beese and T. A. Steitz (1991). EMBO ]. 10, 25-33.)... 

Although the transition states are depicted in accord with the preference rules, the presence of pentacovalent phosphorus is conjecture. 

The chemistry and stereochemistry of the reactions were extensively discussed in Chapter 8, sections El and E3. There is an in-line mechanism that generates a pentacovalent intermediate or transition state, with the attacking nucleophile and leaving group occupying the apical positions of the trigonal bipyramid. 

In the in-line push-pull mechanisms of Rabin and Roberts, the highest energy transition state may be either the pentacovalent intermediate or the alkoxide (hydroxide) state with 02 or 05" deprotonated but not bonded to P. Incipient deprotonation of 02 in an activated state is equivalent. Protonation of X or Y or nearby positive charge could stabilize the pentacovalent intermediate. Removal of either could facilitate formation of the alkoxide in the breakdown of the intermediate. Restoration of the initial state of the enzyme is required in this mechanism and could be rate limiting. In the adjacent (pseudorotation) models of Witzel, Hammes, Usher, or Wang protonation of X or Y would be required to allow one of the two pseudomers to exist. In step 1 this requirement (and thus perhaps a rate limiting process) applies to the attack by 02. Deprotonation would force or facilitate reversal or pseudorotation to... 

The ks curve for C > p hydrolysis is not symmetrical, and a secondary acid pathway has been proposed to explain part of the activity at pH 4.0 (499). Perhaps the protonation of both histidines produces enough polarization of the phosphate to permit direct attack by water or stabilizes the transitional state in the formation of the pentacovalent intermediate. With excess protons present, protonation of the leaving group would be easy. If histidine is not involved as a base, the ks/Km curve should also be distorted according to this view. This description would not seem to fit their model. In any case it would be interesting to see if this acid pathway is different from the normal pathway with respect to in-line vs. adjacent mechanisms. An alternate proposal is that the protonation of the acetate-tris buffer was distorting the curve. Another factor to consider is the chloride behavior as a competitive inhibitor, but this would not affect fcs if it is strictly competitive. 

West (91), amplifying on these results, argued that since the solvolysis is bimolecular it must proceed either through a normal Sn2 bimolecular displacement or involve a rather stable pentacovalent intermediate. Both mechanisms. West believes, must involve a 5-coordinate transition state, and therefore may really be thought of as equivalent. West found that silacyclopentane was 13 times as reactive as diethylmethylsilane and 200 times as reactive as silacydohexane (which could be construed as evidence for I-strain in silacyclopentane). Since this order of reactivity is the same as that found in carbocyclic compounds, it was concluded that similar considerations of energy and entropy of reaction are encountered, a possibility that had also been advanced by Price. 

It should be noted that this transition state does not differ too greatly from that postulated for the alkaline catalyzed reaction by Kaplan and Wilzbach. The latter, however, is formed by the reaction of a solvent molecule with a pentacovalent negative ion formed in a rate controlling step whereas in the other instance, the rate controlling step is the attack of H30+ on the neutral transition complex. Further consideration and reconciliation of these views will obviously be of interest. 

As displayed above there is an anomerization of 44 to 49 due to trans-(2 1)-0- (44 46) and ensuring m-(2—>l)-0- (47- 49) silyl group migration, presumably via pentacovalent silicon intermediates or transition states 45 and 48. 

The immediate product is presumably the epoxide 213 which opens in acetal fashion to give 214 and transfers silicon intramolecularly to give 212. The reaction 214 is formally a 5-endo-tet reaction and would not occur if carbon were being transferred. As silicon is the atom under attack, a pentacovalent intermediate can be formed and the requirement for a linear SN2 transition state no longer applies. 

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