Simple but efficient models for nuclease catalysis

The dianions of hydroxyphosphate diesters (3) designed to have high effective molarities for intramolecular cyclisations are hydrolysed in water at 5OoC with half-lives as short as 40 ms. General acid catalysis is characterised in detail for a methyl ester. An important electrostatic effect, observed for general acids with a suitably positioned second NH+ group, enhances the rate of the loss of methoxide from the dianion by 100fold even in water. The data allow an estimate of lo* 105 for the factor by which protonation to give the neutral acid activates a phosphate diester anion towards attack by a neighbouring OH group. The results define basic requirements for efficient catalysis of the reaction, which is common to many enzymes which process nucleic acids. INTRODUCTION Of the phosphate esters which play a role in living systems, diesters are the most important and the least reactive. The stability towards hydrolysis of DNA is crucial to the conservation of the genetic information, and even RNA, though readily hydrolysed in alkali, is cleaved only very slowly at pH 7 in the absence of a relevant enzyme. This hydrolysis is in fact so slow that good quality data on catalysis of the reaction are difficult to obtain. This in turn makes it difficult to draw firm conclusions about mechanism, and there is conflicting evidence on the status transition state or intermediate of the pentacovalent species involved in such reactions. Two factors have given new impetus to the field. One is the work of Breslow and his co-workers, who measured the very slow hydrolysis of ribonucleoside derivatives (for example, UpU) by a method based on HPLC separation of the products (ref. 1). This work has been criticised (ref. 2), and the data are necessarily of limited accuracy, and in some cases inconsistent with other published data (ref. 3), so cannot support a unique interpretation. But they break new ground, and lead to interesting suggestions for the mechanism of the hydrolysis reaction which deserve careful consideration. The work described in this paper addresses two of these in particular: the details of general acid-base catalysis of the reaction in which a neighbouring OH group attacks the phosphorus centre of a phosphate diester, and the suggestion of Breslow and his co-workers that the substrate for the initial reaction is the phosphoric acid rather than the anion of the phosphate diester. The second important new factor is the explosive growth in the number of enzymes known to 'process' nucleic acids, catalysing various sorts of sequence-modifications. These include recombinases (over 2,000 are now known: ref. 4), topoisomerases (ref. 5) and integrases (ref. 6); and even non-enzymes like ribozymes (ref. 7). All appear to involve a common mechanism by which phosphate transfer takes place by a two step process via a phosphoryl enzyme (or ribozyme) intermediate. The catalytic nucleophilic centre is typically a hydroxyl group, of a nucleotide ribose residue in the case of a ribozyme and of a serine or tyrosine side-chain of the case of the enzyme reactions. In none of these cases does reaction involve the formation of a five687 688 K. N. DALBY, A. J. KIRBY AND F. HOLLFELDER membered ring, as in the ribonuclease reaction (where 1 + 2 is the initial step), so a further question of interest is whether the special structural features of ribonucleotides favour a unique mechanism. (The Breslow mechanism (ref. 1) involves mechanistically unsymmetrical partitioning of a pentacovalent intermediate.) 4 OH P. m'\ 0 O 1 R'O,