Thiol subtilisin

Polgar L. and Bender, M.L. (1966) A new enzyme containing a synthetically formed active site. Thiol-subtilisin. J. A/w. Chem. Soc., 88, 3153-3154. 

Wild-type subtilisin BPN with the mutation Ser — Cys-24 has a kcat value of 59 s 1 and a KM value of 200 fxM with the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide, compared with a rate constant of 1.1 X 10 8 s 1 for its spontaneous hydrolysis under the same conditions. Replacement of Asp-32, His-64, and Ser-221 one at a time by alanine reduced the value of kcat by factors of 3 X 104, 2 X 106, and 2 X 106, respectively. Converting all three to alanine also decreases activity by 2 X 106. The value of KM increases only by a factor of two on all these mutations.34 It is unlikely that the residual activity results from the presence of a small amount of wild-type active site in the thiol mutants... 

With regard to the use of protease in the synthetic mode, the reaction can be carried out using a kinetic or thermodynamic approach. The kinetic approach requires a serine or cysteine protease that forms an acyl-enzyme intermediate, such as trypsin (E.C. 3.4.21.4), a-chymotrypsin (E.C. 3.4.21.1), subtilisin (E.C. 3.4.21.62), or papain (E.C. 3.4.22.2), and the amino donor substrate must be activated as the ester (Scheme 19.27) or amide (not shown). Here the nucleophile R3-NH2 competes with water to form the peptide bond. Besides amines, other nucleophiles such as alcohols or thiols can be used to compete with water to form new esters or thioesters. Reaction conditions such as pH, temperature, and organic solvent modifiers are manipulated to maximize synthesis. Examples of this approach using carboxypeptidase Y (E.C. 3.4.16.5) from baker s yeast have been described.219... 

EndopepUdases (proteinoses) catalyse the hydrolysis of bonds within the peptide chain, forming variously sized cleavage peptides. They can be further subdivided into acidic, neutral and basic endopeptidases. Neutral and basic types can each be divided into Serine proteases (see) and thiol proteinases (see Thiol enzymes). Examples of animal endopeptidases are Pepsin (see). Rennet enzyme (see), Ttypsin (see), Elastase (see). Thrombin (see), Plasmin (see) and Renin (see). For examples of plant and bacterial endopeptidases, see Papain, Subtilisin, Bromelain. Endopeptidases have also been isolated from yeast and fungi. 

Thermolysin (EC 3.4.24.4) a heat-stable, zinc- and calcium-containing neutral protease, M, 37,500, from Bacillus thermoproteolyticus, with a substrate specificity similar to that of Subtilisin (see). After one hour at 80 °C, T. still has 50% original activity. This high heat stability of T. is attributed to the large number of hydrophobic regions and the presence of four bound calcium ions, which serve in place of disulfide bridges (T. contains no disulfide bridges) to maintain the compact shape of the molecule. T. is neither a thiol nor a serine enzyme. 

Enzymes that hydrolyze amide and ester bonds may be divided into three classes (1) those requiring a thiol group for activity, such as papain, ficin, and other plant enzymes (2) those inhibited by diisopropylphosphorofluo-ridate (DFP), such as a-chymotrypin, trypsin, subtilisin, cholinesterase, and thrombin (3) those that require a metal ion for activity. This last class includes dipeptidases, and exopeptidases such as carboxypeptidase and leucine aminopeptidase. The metal ion is involved in the stabilization of the tetrahedral intermediate (refer to Section 4.4.1). 

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