The active site of esterases

Abstract

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...

Acetylcholinesterase in the fast extraocular muscle of the mouse by light and electron microscope autoradiography.

“…The conditions were also designed to eliminate any significant effect of "ageing" on the quantitative results by keeping the time lapse between the DFP and the 2-PAM incubation to <1 h. ("Ageing" is a process whereby dealkylation of the organophosphorous inhibited enzymes occurs with time and prevents reactivation by 2-PAM. The accumulated evidence from several studies [5,9,11,14,21,22,23] leads to the conclusion that the half-life for ageing of AChe inactivated with DFP is longer than for butyrylcholinesterase (BuChe), is on the order of 10-35 h at room temperature, and is temperature-dependent. )…”

Affinity Labeling of the Active Sites of Antibodies to the 2,4-Dinitrophenyl Hapten*

“…Eisen, 1962;Day et al, 1962;Nisonoff and Pressman, 1958). The question arises: do those Ab sites which are most rapidly labeled with a HpX reagent show the largest K , values for the reversible reaction with Hp?…”

Purification and Properties of the Inducible Cholinesterase of Pseudomonas Fluorescens (Goldstein)

“…Kinetic studies (17,18) and direct chemical evidence (19) support the hypothesis that the essential basic group of esterases (pK, 6-7) is the in~idazole ring of histidine. The consistent isolation s f labelled serine phosphate derivatives from esterases after their inhibition by organophosphorus coinpounds (20) has provided strong evidence that serine is associated with the active site. On the basis of his pH-activity data and observations with T E P P and BFP, Fitch (4) concluded that neither histidine nor serine were likely to be of importance in the activities of the bacterial cholinesterase.…”