THE CHEMISTRY AND EVOLUTION OF THE CATALYTIC STRUCTURES IN SERINE HYDROLASES

G G Dodson

Department of Chemistry, University of York, YORK YO1 5DD UK

The speed and specificity of enzyme reactions are amongst the most remarkable features of biological chemistry. These molecules' chemistry and kinetics have been studied extensively but it was not until the crystal structures were determined that the structural organisation responsible for the catalytic potentiation, for the stabilisation of the transition states and for the specificity could be analysed. The crystal structures of hundreds of enzymes have now been determined, often as complexes with analogues of intermediate states in the chemical reactions. thus there is a reasonably accurate picture of the structural events in many enzyme mechanisms though there remain serious shortcomings in understanding the chemical and electronic contributions of the individual residues. For historical reasons, and because of their biological importance, there has been a great deal of research into the serine hydrolases such as the serine proteases trypsin and subtilisin, and the members of their families.

The active sites of trypsin and subtilisin contain a so-called catalytic triad asp : his : ser, in which the his and asp act together to activate the seryl OH into a powerful nucleophilic centre. It was shown about 25 years ago that there is an essentially identical structural arrangement of the active atoms in the two independently evolved serine proteases chymotrypsin and subtilisin. This was an important example of chemical constraints acting on the evolutionary selection of the catalytic residues. Over the last period a number of new families of serine (or threonine) hydrolases have been identified. These enzymes cleave esters, amides, b-lactamases and peptides with the same nucleophilic mechanism as seen in the serine protease. Crystallographic analysis has also demonstrated the existence of evolutionary relationships that are not detectable from sequence comparisons alone. This has revealed new and deeper patterns of molecular evolution based on the 3 dimensional structure of the main chain residue containing the nucleophilic atom. The variation in the catalytic structures reveals nonetheless that the chemical requirements for catalytic enhancement have enforced a common organisation which was generally acid : base :ser(thr), corresponding in serine proteases to asp : his : ser. There are exceptions to this which provide clues to the role of the component catalytic groups in activation and to the differing stereochemistry of the scissile bonds.

Recently a new hydrolase family has been identified in which the active residue (ser, thr or cys) is at the N terminus and incorporates the a-amino group in the catalytic structure explaining their naming as Ntn hydrolases. This structural economy is accompanied by a characteristic tertiary structure, revealed by the crystallographic studies. Most interestingly Ntn hydrolases are usually synthesised as an inactive precursor which is activated by autocatalytic removal of peptide to create the catalytically active N terminus. These enzymes prove to have a wide range of substrates involved in a diverse range of biochemical pathways. The variation in the sequences of Ntn hydrolases, which extends to the catalytic structure itself is striking. It illustrates the immense potential for chemical reactions residing within protein surfaces which on an appropriate main chain framework can vary hugely in response to local cellular demands and still meet the chemical and structural demands of catalysis.