Diisopropyl chymotrypsin

Pohl (1968a,b, 1969, 1972a,b) developed a simple method (which was in fact a slow temperature jump method) to follow reversible thermal unfolding of globular proteins. Reversible denaturation of trypsin, chymotrypsin, and chemically modified derivatives (i.e., anthranyloyl chymotrypsin and diisopropyl chymotrypsin) and ribonuclease A were studied. The thermal denaturation was followed for different pH values. From the data obtained for chymotrypsin as well as for trypsin, Pohl (1968a,b) concluded that there was a two-state process. This conclusion was supported by the following arguments ... 

Such an intermediate is known to be formed in reactions catalyzed by trypsin, chymotrypsin, thrombin, other enzymes of the blood-clotting cascade (except angiotensinconverting enzyme, which is an aspartic protease). An acyl-serine intermediate is also formed in the acetylcholinesterase reaction. The active site serine of this enzyme and the serine proteases can be alkylated by diisopropyl-fluorophosphate. See also Active Site Titration... 

Mazur and Bodansky (1946) found that diisopropyl fluorophosphate (DFP) irreversibly inhibits acetylcholine esterase. In particular, in 1949 Jansen, Balls, and their collaborators demonstrated the stoichiometric reaction of DFP with chymotrypsin (Jansen et al., 1949a,b Aldridge, 1950). 

The mammalian serine proteases have a common tertiary structure as well as a common function. The enzymes are so called because they have a uniquely reactive serine residue that reacts irreversibly with organophosphates such as diisopropyl fluorophosphate. The major pancreatic enzymes—trypsin, chymotrypsin, and elastase—are kinetically very similar, catalyzing the hydrolysis of peptides... 

The preceding experiments prove that there is an intermediate on the reaction pathway in each case, the measured rate constants for the formation and decay of the intermediate are at least as high as the value of kcat for the hydrolysis of the ester in the steady state. They do not, however, prove what the intermediate is. The evidence for covalent modification of Ser-195 of the enzyme stems from the early experiments on the irreversible inhibition of the enzyme by organo-phosphates such as diisopropyl fluorophosphate the inhibited protein was subjected to partial hydrolysis, and the peptide containing the phosphate ester was isolated and shown to be esterified on Ser-195.1516 The ultimate characterization of acylenzymes has come from x-ray diffraction studies of nonspecific acylenzymes at low pH, where they are stable (e.g., indolylacryloyl-chymotrypsin),17 and of specific acylenzymes at subzero temperatures and at low pH.18 When stable solutions of acylenzymes are restored to conditions under which they are unstable, they are found to react at the required rate. These experiments thus prove that the acylenzyme does occur on the reaction pathway. They do not rule out, however, the possibility that there are further intermediates. For example, they do not rule out an initial acylation on His-57 followed by rapid intramolecular transfer. Evidence concerning this and any other hypothetical intermediates must come from additional kinetic experiments and examination of the crystal structure of the enzyme. 

Proteins. Bovine insulin, a-chymotrypsin and ribonuclease were purchased from Sigma Chemical Company. Inactivated diisopropyl-phosphoryl(DIP)-a-chymotrypsin was prepared by incubation with diisopropylfluorophosphate(DFP) (25). 

Enzyme inhibitors are often poisonous. For example, diisopropyl-fluorophosphate is a nerve poison because the enzyme acetylcholinesterase has a reactive site serine. Chymotrypsin and acetylcholinesterase are both members of the class of enzymes known as serine esterases, which are all inhibited by diisopropylfluorophosphate. 

The nature of the active center of chymotrypsin and other hydrolytic enzymes (in general, esterases) has been explored with a specific type of inhibitor, dialkyl phosphate anhydrides, such as diisopropyl-fluorophosphate (DFP). These compounds react with chymotrypsin to add one, and only one, phosphorus per molecule and form a completely inactive compound. The inactivated molecule yields phosphoserine on... 

The mechanism of the action of acetylcholinesterase purified from the electric organs of Electrophorus electricus involves the attraction of the positively charged nitrogen of acetylcholine to an anionic site on the enzyme and cleavage of the substrate at an esteratic site of a nucleophilic character. The irreversible inhibition by the alkyl phosphates, tetraethyl pyrophosphate (TEPP) and diisopropyl-fluorophosphate (DFP) may be due to phosphorylation of the nucleophilic esteratic site. The phosphorylation by DFP of the phenolic hydroxyl group of free tyrosine has been demonstrated by Ashbolt and Rydon. Chymotrypsin and citrus fruit acetylesterase are also inhibited by DFP and TEPP. ... 

Hachimori, Y., K. Kurihara, H. Horinishi, A. Matsushima, and K. Shibata States of Amino Acid Residues in Proteins. VIII. Tyrosine, Histidine, and Tryptophan Residues in Chymotrypsin in the Presence of Substrate and in Diisopropyl-phosphorylchymotrypsin. Biochim. Biophys. Acta 105, 167-177 (1965). 

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