Peptide hydrolysis rate

Field studies point in a similar direction field comparisons of peptide hydrolysis rates and amino acid turnover in coastal sediments showed that amino acid production could exceed uptake by a factor of approximately 8 (Pantoja and Lee, 1999). A comparison of potential enzyme activities and sedimentary amino acid and carbohydrate inventories in sediments from the Ross Sea also showed that potential hydrolysis rates on time scales of hours should in theory rapidly deplete sedimentary amino acid and carbohydrate inventories (Fabiano and Danovaro, 1998). In deep-sea sediments, Poremba (1995) also found that potential enzyme activities in theory could exceed total sedimentary carbon input by a factor of 200. Finally, Smith et al. s (1992) investigation of potential hydrolysis rates and amino acid uptake in marine snow demonstrated that the particle-associated bacteria were potentially producing amino acids far in excess of their own carbon demand. 

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... 

If prebiotic peptides and/or proteins were in fact initially formed in aqueous solution (the hypothesis of biogenesis in the primeval ocean ), the energy problems referred to above would have needed to be solved in order for peptide synthesis to occur. As discussed in Sect. 5.3, there is some initial experimental evidence indicating that the formation of peptide bonds in aqueous media is possible. An important criterion for the evolutionary development of biomolecules is their stability in the aqueous phase. The half-life of a peptide bond in pure water at room temperature is about seven years. The stability of the peptide bond towards cleavage by aggressive compounds was studied by Synge (1945). The following relative hydrolysis rates were determined experimentally, with the relative rate of hydrolysis for the dipeptide Gly-Gly set equal to unity ... 

Peptide hydrolysis by platinum(II) (436) and palladium(II) complexes (437). In the latter case there is selective hydrolysis of the unactivated peptide bond in iV-acetylated L-histidylglycine the hydrolysis rate depends on the steric bulk of the catalyst. 

Hence, by the principle of microscopic reversibility, the reverse reaction (the hydrolysis of peptides by the acylenzyme mechanism) must also occur. The question is whether or not this reaction is rapid enough to account for the observed hydrolysis rate. This can be answered by measuring (kcJKM)s for the synthesis of a peptide by the acylenzyme route, and for the hydrolysis of the peptide (kcJKM)u for the hydrolytic reaction can then be calculated from the Haldane equation,... 

Effective concentration 65-72 entropy and 68-72 in general-acid-base catalysis 66 in nucleophilic catalysis 66 Elastase 26-30, 40 acylenzyme 27, 40 binding energies of subsites 356, 357 binding site 26-30 kinetic constants for peptide hydrolysis 357 specificity 27 Electrophiles 276 Electrophilic catalysis 61 metal ions 74-77 pyridoxal phosphate 79-82 Schiff bases 77-82 thiamine pyrophosphate 82-84 Electrostatic catalysis 61, 73, 74,498 Electrostatic effects on enzyme-substrate association rates 159-161... 

Trypsin was named more than 100 years ago. It and chymotrypsin were among the first enzymes to be crystallized, have their amino acid sequences determined, and have their three-dimensional structure outlined by x-ray diffraction. Furthermore, both enzymes hydrolyze not only proteins and peptides but a variety of synthetic esters, amides, and anhydrides whose hydrolysis rates can be measured conveniently, precisely and, in some instances, extremely rapidly. As a result, few enzymes have received more attention from those concerned with enzyme kinetics and reaction mechanisms. The techniques developed by the pioneers in these various fields have enabled other serine proteases to be characterized rapidly, and the literature on this group of enzymes has become immense. It might be concluded that knowledge of serine proteases is approaching completeness and that little remains but to fill in minor details. 

A study conducted in deep waters of the North Atlantic found no preferential microbial utilization of L- or D-amino acids during uptake experiments (Perez et al., 2003) yet, the presence of D-amino acids may retard the hydrolysis rate of peptides. But D/L-amino acid ratios in DOM and HWMDOM rarely exceed 1 even in the deep ocean (some surface ocean samples analyzed by Perez et al. (2003) did show values >1 for d/l-aspartic acid). The ratio could approach or exceed 1 if peptides containing D-amino acids (e.g., peptidoglycan, which contains only a few amino acids) were preferentially preserved with depth. Instead, near constant d/l ratios... 

Figure 3 Complex formation between trypsin and soybean trypsin inhibitor exemplifies the mechanism of Kunitz-type inhibitors (PDB 1AVW). Greatly reduced peptide bond hydrolysis rates lead to inhibition.

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. 

Figure 7. Liposome-assisted catalysis. (A) Dependency of the initial hydrolysis rate of C16-O Np (nitrophenyl-pamitate) catalyzed by 1 mM carbobenzoxy-Phe-His-Leu-OH on the substrate concentration, in 0.05 M borate buffer pH 8.5. The filled circles are relative to the self-hydrolysis (no peptide, no liposomes). Open triangles are without liposomes, open squares with liposomes. (B) The pseudo-enzymatic turnover of the catalytically active liposomes. The catalytic activity results primarily from the binding (and solubilization) of a very hydrophobic histidin-containing peptide and the very hydrophobic substrate.

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