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This paper presents data from our laboratory pertaining to the chemical structure of the active site of a group of esterases. We attempted to frame these into a general pattern in agreement with wellestablished knowledge in this field. First we should specify that the term "active site" will refer to those regions of the enzyme surface where the substrate is localized and activated during the enzymic action.Knowledge of the structure of the active site of esterases is based on indirect evidence mostly obtained by kinetic methods and on direct evidence; the latter usually results from chemical analysis of components of the enzyme protein. The former category will be dealt with only summarily because ( 1 ) it has already received much attention by reviewers, (2) it tends to produce results that defy straightforward interpretation, and ( 3 ) our personal experience has been limited to chemical methods. Therefore, the evidence based on the appIication of chemical methods will weigh most heavily in our interpretation of the experimental data.Among the chemical methods, those involving a specific reaction of the active site of an enzyme followed by the analysis of the groups involved seem the most direct and attractive. Our work was based on such a specific reaction; viz., the property of many esterases to react with diisopropyl fluorophosphate (DFP) to form an enzymically inactive compound. This compound was broken down by proteolytic enzymes, and the structure that carried the diisopropylphosphoryl (DP) residue was analyzed. A rigorous requirement for the validity of the method is the certainty that DFP reacts specifically with the active site. It is now generally agreed that this is so for the following reasons.The enzymes concerned were inhibited by low concentrations of DFP. Where the molecular weight of the enzyme was known (in trypsin and chymotrypsin), 1 mole of enzyme reacted with 1 mole of DFP to give complete inhibition; correspondingly, when inhibition was only partial, its degree was always linearly related to the amount of DP bound (Balls and Jansen, '52). Other evidence is provided by the well-known ability of substrates to prevent the inhibition by DFP, indicating competition for a common active site. Finally, it is now generally accepted that the inhibition by DFP involves a permanent phosphorylation of the active site analogous to the transient acylation of this site in the course of normal substrate hydrolysis.Our first results based on this approach (Cohen, . . . Jansz, '55) demonstrated that a number of DFP-sensitive enzymes carry a very similar structure reacting with DFP. We suggested that this common structure (the B group) is cIosely associated with the general property of enzymic hydrolysis. The substrate specificity of the enzyme, however, would be determined by additional chemical groups on or near the active site. This B group will necessarily consist of amino acids although structures resulting from interaction of amino acid side chains may occur. Results of the continuation of this work w...

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