Reverse protease reaction

Fragment condensation of peptides corresponds to a reverse protease reaction -peptide synthesis instead of cleavage - and this is well known in the hterature as well. In fact, proteases have been used extensively for peptide coupling (Jakubke etal, 1985 1996 Jakubke, 1987 1995 Luisi etal, 1977b). This work has shown that even small proteins can be synthesized by block-wise enzymatic couphng (see also Kullmann, 1987, and, for some more recent developments, Celovsky and Bordusa, 2000). 

Transition-state inhibitors stably mimic the transition state of the enzymatic reaction, and thereby interact with the substrate-bin-ding and catalytic machinery of the enzyme in a low-energy conformation. Transition-state analogs are competitive, reversible inhibitors, although some have extremely low Kj s and very slow off-rates. All proteases activate a nucleophile to attack a carbonyl, which leads to the formation of a tetrahedral intermediate that then collapses to form the enzyme products—two peptides. Thus, synthetic small molecules that mimic the tetrahedral intermediate of the protease reaction are attractive transition-state analogs. A classic class of protease transition-state inhibitors uses a boronic acid scaffold (4, 10). Boronic acid adopts a stable tetrahedral conformation in the protease active site that is resistant to nucleophilic attack. Boronic acid inhibitors, which are derivatized with different specificity elements, have been developed against every class of protease... 

Since the beginning of enzyme catalysis in microemulsions in the late 1970s, several biocatalytic transformations of various hydrophilic and hydrophobic substrates have been demonstrated. Examples include reverse hydrolytic reactions such as peptide synthesis [44], synthesis of esters through esterification and transesterification reactions [42,45-48], resolution of racemic amino acids [49], oxidation and reduction of steroids and terpenes [50,51], electron-transfer reactions, [52], production of hydrogen [53], and synthesis of phenolic and aromatic amine polymers [54]. Isolated enzymes including various hydrolytic enzymes (proteases, lipases, esterases, glucosidases), oxidoreductases, as well as multienzyme systems [52], were anployed. 

Drugs are also used to inhibit the enzymatic reactions of foreign pathogens that enter the human body. An example is the use of reverse transcriptase inhibitor and protease inhibitor for combating the human immunodeficiency virus (HIV), as shown in Exhibit 2.12. Some new inhibitors are used to block HIV from attaching to the human cell, CD4, thus stopping replication and infection of other cells, as presented in Exhibit 2.13. 

As discussed above, proteases are peptide bond hydrolases and act as catalysts in this reaction. Consequently, as catalysts they also have the potential to catalyze the reverse reaction, the formation of a peptide bond. Peptide synthesis with proteases can occur via one of two routes either in an equilibrium controlled or a kinetically controlled manner 60). In the kinetically controlled process, the enzyme acts as a transferase. The protease catalyzes the transfer of an acyl group to a nucleophile. This requires an activated substrate preferably in the form of an ester and a protected P carboxyl group. This process occurs through an acyl covalent intermediate. Hence, for kineticmly controlled reactions the eii me must go through an acyl intermediate in its mechanism and thus only serine and cysteine proteases are of use. In equilibrium controlled synthesis, the enzyme serves omy to expedite the rate at which the equilibrium is reached, however, the position of the equilibrium is unaffected by the protease. 

Selected entries from Methods in Enzymology [vol, page(s)] Association constant determination, 259, 444-445 buoyant mass determination, 259, 432-433, 438, 441, 443, 444 cell handling, 259, 436-437 centerpiece selection, 259, 433-434, 436 centrifuge operation, 259, 437-438 concentration distribution, 259, 431 equilibration time, estimation, 259, 438-439 molecular weight calculation, 259, 431-432, 444 nonlinear least-squares analysis of primary data, 259, 449-451 oligomerization state of proteins [determination, 259, 439-441, 443 heterogeneous association, 259, 447-448 reversibility of association, 259, 445-447] optical systems, 259, 434-435 protein denaturants, 259, 439-440 retroviral protease, analysis, 241, 123-124 sample preparation, 259, 435-436 second virial coefficient [determination, 259, 443, 448-449 nonideality contribution, 259, 448-449] sensitivity, 259, 427 stoichiometry of reaction, determination, 259, 444-445 terms and symbols, 259, 429-431 thermodynamic parameter determination, 259, 427, 443-444, 449-451. 

Other major untoward reactions are the result of rifampin s ability to induce hepatic cytochrome P-450 enzymes, leading to an increased metabolism of many drugs this action has especially complicated the treatment of tuberculosis in HIV-infected patients whose regimen includes protease inhibitors and nonnucleoside reverse transcriptase. Since rifabutin has relatively little of these effects, it is commonly substituted for rifampin in the treatment of tuberculosis in HIV-infected patients. 

Formation of an amide bond (peptide bond) will take place if an amine and not an alcohol attacks the acyl enzyme. If an amino acid (acid protected) is used, reactions can be continued to form oligo peptides. If an ester is used the process will be a kinetically controlled aminolysis. If an amino acid (amino protected) is used it will be reversed hydrolysis and if it is a protected amide or peptide it will be transpeptidation. Both of the latter methods are thermodynamically controlled. However, synthesis of peptides using biocatalytic methods (esterase, lipase or protease) is only of limited importance for two reasons. Synthesis by either of the above mentioned biocatalytic methods will take place in low water media and low solubility of peptides with more than 2-3 amino acids limits their value. Secondly, there are well developed non-biocatalytic methods for peptide synthesis. For small quantities the automated Merrifield method works well. 

The hormone-hke peptide incretin stimulates the release of insuhn by a feedback process that involves cleaving the molecule to an inactive form. The protease enzyme dipeptidal peptidase (DPP) in turn cleaves incretin, in effect inactivating this enzyme. Inhibition of DPP consequently extends the action of incretin. This inhibition thus prevents the increased levels of blood glucose that mark diabetes. The protease inhibitor vidagliptin, which is modeled in part on the terminal sequence in DPP, has been found to sustain levels of insulin in Type II diabetics. The inhibition is apparently reversible in spite of the presence in the structure of the relatively reactive a-aminonitrile function. Construction of one intermediate in the convergent synthesis comprises the reaction of amino adamantamine (21-1) with a mixture of nitric and... 

In nature, the main purpose of proteases is to cleave peptide bonds. As the peptide and its amino acid components are in equilibrium, and as catalysts always catalyze both directions of a reversible reaction, the same reaction in principle can be used to synthesize peptide bonds. 

When one is using proteases in a direct reversal of their normal hydrolytic function, the equilibrium position is very important in limiting the attainable yield in equilibrium-controlled enzymatic peptide synthesis. If both reactants and products are largely undissolved in the reaction medium as suspended solids, thermodynamic analysis of such a system shows the reaction will proceed until at least one reactant has dissolved completely, towards either products or reactants ( switchlike behavior). In case of a favorable equilibrium for synthesis, the yield is maximized in the solvent of least solubility for the starting materials (Hailing, 1995). Thermolysin-catalyzed reactions ofX-Phe-OH (X = formyl, Ac, Z) with Leu-NH2 yielded X-Phe-Leu-NH2 with equilibrium yields > 90% over a range of solvents. Some predictions, such as a linear decrease in yield with the reciprocal of the initial reactant concentrations, could be verified (Hailing, 1995). 

Hi) Enzymatic Esterifications. A major alternative to the classical basic catalysis is the use of enzymes for esterification, in particular with proteases and lipases.110 112 To make these enzymes, which normally hydrolyze amide or ester linkages, work in the reverse direction of esterification, the reactions have to be performed in organic media, with only the small amount of water necessary to preserve their active conformation. In such reactions, the difficulty is to find those conditions of solvent and temperature compatible with both the solubility of the substrates and the stability and the activity of the enzyme.113,114 In the case of sucrose (Scheme 9), most proteases lead selectively to monoesters at position p nl-ii5,ii6 Ypggg reactions are often performed in DMF, but examples in Me2SO, which is much less toxic, have also been reported, despite the ability... 

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