Acid-catalyzed racemization protein amino acids

H Frank, W Woiwode, G Nicholson, E Bayer. Determination of the rate of acidic catalyzed racemization of protein amino acids. Liebigs Ann Chem 354, 1981. 

Preparative Methods substituted 2,3-methanoamino acids are difficult to prepare. Unfortunately, most of the reported syntheses give racemic materials whereas stereochemically pure compounds are required for studies of cyclopropane-based peptidomimetics. The only 2,3-methanologs of protein amino acids prepared in optically active form are ( )- and (Z)-cyclo-Phe and -Tyr, all four stereoisomers of cyc/o-Met, (Z)-cyclo-Arg and (25,35)-(Z)-cyc/o-Trp, although several routes to enantio-enriched 2,3-methanologs of simple nonproteogenic amino acids have been reported. " The most practical synthesis of the title compound is that based on a diastereoselective, rhodium-catalyzed cyclopropanation reaction. ... 

It is remarkable that virtually all amino acid residues in proteins are L stereoisomers. When chiral compounds are formed by ordinary chemical reactions, the result is a racemic mixture of d and l isomers, which are difficult for a chemist to distinguish and separate. But to a living system, D and L isomers are as different as the right hand and the left. The formation of stable, repeating substructures in proteins (Chapter 4) generally requires that their constituent amino acids be of one stereochemical series. Cells are able to specifically synthesize the l isomers of amino acids because the active sites of enzymes are asymmetric, causing the reactions they catalyze to be stereospecific. 

The amide bonds in peptides and proteins can be hydrolyzed in strong acid or base Treatment of a peptide or protein under either of these conditions yields a mixture of the constituent amino acids. Neither acid- nor base-catalyzed hydrolysis of a protein leads to ideal results because both tend to destroy some constituent ammo acids. Acid-catalyzed hydrolysis destroys tryptophan and cysteine, causes some loss of serine and threonine, and converts asparagine and glutamine to aspartic acid and glutamic acid, respectively. Base-catalyzed hydrolysis leads to destruction of serine, threonine, cysteine, and cystine and also results in racemization of the free amino acids. Because acid-catalyzed hydrolysis is less destructive, it is often the method of choice. The hydrolysis procedure consists of dissolving the protein sample in aqueous acid, usually 6 M HC1, and heating the solution in a sealed, evacuated vial at 100°C for 12 to 24 hours. 

Recently a simplified process was developed for incorporating l-methionine directly into soy proteins during the papain-catalyzed hydrolysis (21). The covalent attachment of the amino acid requires a very high concentration of protein and occurs through the formation of an acyl-enzyme intermediate and its subsequent aminolysis by the methionine ester added in the medium. From a practical point of view, the main advantage of enzymatic incorporation of amino acids into food proteins, in comparison with chemical methods, probably lies in the fact that racemic amino acid esters such as D,L-methionine ethyl ester can be used since just the L-form of the racemate is used by the stereospecific proteases. On the other hand, papain-catalyzed polymerization of L-methio-nine, which may occur at low protein concentration (39), will result in a loss of methionine because of the formation of insoluble polyamino acid chains greater than 7 units long. 

Heat and alkaline treatments have been known since the early part of the century to raoemize amino acid residues in proteins (1,2,). Dakin and Dudley (3) also studied digestibility of casein in vitro and in vivo after hydroxide treatment. Heating casein with 0.5 N NaOH at 37° for about 30 days completely prevented enzymatic hydrolysis and intestinal absorption when the treated casein was fed to a dog. The kinetics of base-catalyzed racemization of proteins was investigated by Levene and Bass (4-6). In these early studies, the extent of racemization was measured by changes in optical rotation. 

Pyridoxal 5 -phosphate dependent enzymes constitute an important class of proteins involved predominately in amino acid metabolism. The PLP-cofactor is capable of catalyzing a variety of reactions at the a-, [3-, and/or y-carbons of amino acid substrates. These reactions include tranamination, racemization, decarboxylation, and aldoyltic cleavage reactions at the a-carbon and elimina-tion/substitution reactions at either the 3-, or y-position of the amino acid substrate (67-74) The chemical properties of the cofactor (67-71) are responsible for the great diversity of reactions catalyzed by PLP, while reaction specificity is ultimately determined by the active site environment imposed by the surrounding apo-protein to which the cofactor is covalendy bound (69). 

The general arguments about the antiquity of cofactors apply to PLP. The nonenzymatic synthesis of pyridoxal under prebiotic conditions is considered possible, whereas the presence of a 5 phosphate group could hint to an ancestral attachment of the cofactor to RNA molecules. " Furthermore, there are specific grounds to assume that PLP arrived on the evolutionary scene before the emergence of proteins. In fact, in current metabolism, PLP-dependent enzymes play a central role in the synthesis and interconversion of amino acids, and thus they are closely related to protein biosynthesis. In an early phase of biotic evolution, free PLP could have played many of the roles now fulfilled by PLP-dependent enzymes, since the cofactor by itself can catalyze (albeit at a low rate) reactions such as amino acid transaminations, racemizations, decarboxylations, and eliminations. " This suggests that the appearance of PLP may have preceded (and somehow eased) the transition from primitive RNA-based life forms to more modern organisms dependent on proteins. 

Base-catalyzed racemization reactions may occur in any of the amino acids except achiral glycine (Gly) to yield residues in proteins with mixtures of L- and D-configurations. The a-methine hydrogen is removed to form a carbanion intermediate (Fig. 6.26b). The degree of stabilization of this intermediate controls the rate of this reaction. Racemization generally alters the proteins physicochemical properties and biological activity. Also, racemization generates... 

In summary, alkali treatment of casein catalyzes racemization of optically active amino acids. Factors that influence racemization include pH, temperature, and time of treatment. Further studies are desirable to assess (a) how these factors operate in structurally different proteins and (b) the presence of D-amino acids in foods and feeds. (See Table 12 for some preliminary findings). 

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