Peptides hydrolysis mechanism

The mechaiusm by which metalloproteinases execute catalysis has been of interest for many years. Most studies focused on carboxypeptidase A and thermolysin-like proteases for which extensive stmctural, chemical, and biochemical data are available. The first peptide hydrolysis mechanisms to be proposed... 

Antonczak S, Monard G, Ruiz-Lopez MF, Rivail JL. Insights in peptide hydrolysis mechanism by thermolysin a theoretical QM/MM study. J Mol Model 2000 6 527-538. 

By changing Ser 221 in subtilisin to Ala the reaction rate (both kcat and kcat/Km) is reduced by a factor of about 10 compared with the wild-type enzyme. The Km value and, by inference, the initial binding of substrate are essentially unchanged. This mutation prevents formation of the covalent bond with the substrate and therefore abolishes the reaction mechanism outlined in Figure 11.5. When the Ser 221 to Ala mutant is further mutated by changes of His 64 to Ala or Asp 32 to Ala or both, as expected there is no effect on the catalytic reaction rate, since the reaction mechanism that involves the catalytic triad is no longer in operation. However, the enzyme still has an appreciable catalytic effect peptide hydrolysis is still about 10 -10 times the nonenzymatic rate. Whatever the reaction mechanism... 

Figure 12.5 Zinc-hydroxide reaction mechanism for peptide hydrolysis by carboxypeptidase A. (Reprinted with permission from Lipscomb and Strater, 1996. Copyright (1996) American Chemical Society.)...

In Sect. 6.3, we examined, in some detail, the chemical mechanisms of peptide hydrolysis and showed that, while all are relevant in a pharmaceutical context of production and storage, some are fast enough to shorten the half-life and duration of action of some peptides in the body. However, enzymatic reactions of hydrolysis play a much more important role than non-enzymatic ones in the metabolic degradation of peptides [7] [14][138-141], as discussed in the remainder of this chapter. 

The most extensively studied reactions of coordinated amino acid derivatives are those involving nucleophilic attack at the carbonyl group. These aspects, as well as some of those already covered in the previous section, have been reviewed.335-337 Mechanistic aspects of these reactions have also been discussed in Chapter 7.4. The emphasis in this section will be on the synthetic value of stoichiometric reactions of this type. The two most important synthetic processes are peptide hydrolysis and peptide synthesis, both involving the same mechanism. 

A number of complexes of the general type (CoN4(OH)(OH2)]2+ (N4 = a system of four nitrogen donors) stoichiometrically cleave the N-terminal amino acid from di-or tri-peptides. Reactions have been described for N4 = en2,164-165 trien166 167 and tren.168,186 In the case of trien complexes, the proposed mechanism for peptide hydrolysis is shown in Scheme 7. Hydrolysis can occur by two pathways (a) attack by external hydroxide on the O-bonded chelated peptide, and (b) intramolecular attack of coordinated hydroxide on the N-bonded peptide. 

In summary, therefore, it appears that the mechanism of peptide hydrolysis involves the attack of a zinc-bound hydroxide group whose formation is promoted by Glu-270, probably with the formation of a six-coordinate intermediate through binding of substrate. 

The stereochemical aspects of peptide hydrolysis catalyzed by chymotrypsin and related serine proteases has been recently analyzed with respect to requirements for stereoelectronic control of bond cleavage and this analysis has led to a much more complete understanding of the reaction mechanism (9-14). 

N Strater, L Sun, ER Kantrowitz, WN Lipscomb (1999) A bicarbonate ion as a general base in the mechanism of peptide hydrolysis by dizinc leucine aminopeptidase, Proc Natl Acad Sci USA 96 11151-11155... 

Reaction Mechanisms of Metalloproteinase-Mediated Peptide Hydrolysis... 

Here we will discuss the progress achieved so far in our mechanistic understanding of peptide hydrolysis reactions by metalloproteinases. We will focus on the need for comparative structure-function analyses of individual metalloproteinases to reveal the reaction mechanisms. Finally, we will describe new frontiers in the biophysics and chemistry of metalloproteinases and the application of such studies to reveal directly reaction mechanisms and their relevance to dmg discovery. 

Figure 9.8. Peptide Hydrolysis by Chymotrypsin. The mechanism of peptide hydrolysis illustrates the principles of covalent and acid-base catalysis. The dashed green lines indicate favorable interactions between the negatively charged aspartate residue and the positively charged histidine residue, which make the histidine residue a more powerful base.

Proteins are hydrolyzed very slowly with storage in water at neutral pH. However, addition of proteases can increase the rate of hydrolysis about 10 billion times over the spontaneous rate. The chymotrypsin mechanism depicted in Figure 2.53 is shared by trypsin and elastase. These three proteases are members of a family called the serine proteases (named after Ser 195), Carboxypeptidase Aand pepsin catalyze peptide hydrolysis by different mechanisms and are not part of this family. 

Simplified mechanisms of peptide hydrolysis and glycogen phosphorolysis are outlined as follows ... 

Figure 1.24 Mechanism of peptide hydrolysis by a serine protease and enzyme inhibition by forming stable tetrahedral intermediate.

Aminopeptidases are counterparts to carboxypeptidases, removing N-terminal amino acids. However, unlike the carboxypeptidases, they contain dinuclear zinc sites. They fall into two groups, the first of which includes the leucine aminopeptidase from bovine lens, while the second includes the leucine aminopeptidases AAP from Aeromonas proteolytica and SAP from Streptomyces griseus (Figure 12.15). The mechanism of the AAP enzyme has been well studied, and may well represent a general catalytic mechanism for peptide hydrolysis by metal-lopeptidases with a cocatalytic active site.ki. 

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