Base-catalyzed racemization, protein

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. 

Fig. 6.26. Chemical instability of protein biopharmaceuticals, (a) Hydrolysis, (b) Base-catalyzed racemization. (c) P-Elimination.

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... 

Treatment of proteins with alkali will produce many changes, beside loss of optical activity, and the study of base-catalyzed racemization of proteins has not yielded the expected information on protein structure. The many data obtained in this work cannot at the present time be usefully interpreted however, the easy racemization of proteins by alkali and the similar behavior of polypeptides fits into our general picture of protein structure and of the mechanism of racemization as a whole. 

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. 

Mandelate racemase, another pertinent example, catalyzes the kinetically and thermodynamically unfavorable a-carbon proton abstraction. Bearne and Wolfenden measured deuterium incorporation rates into the a-posi-tion of mandelate and the rate of (i )-mandelate racemi-zation upon incubation at elevated temperatures. From an Arrhenius plot, they obtained a for racemization and deuterium exchange rate was estimated to be around 35 kcal/mol at 25°C under neutral conditions. The magnitude of the latter indicated mandelate racemase achieves the remarkable rate enhancement of 1.7 X 10, and a level of transition state affinity (K x = 2 X 10 M). These investigators also estimated the effective concentrations of the catalytic side chains in the native protein for Lys-166, the effective concentration was 622 M for His-297, they obtained a value 3 X 10 M and for Glu-317, the value was 3 X 10 M. The authors state that their observations are consistent with the idea that general acid-general base catalysis is efficient mode of catalysis when enzyme s structure is optimally complementary with their substrates in the transition-state. See Reference Reaction Catalytic Enhancement... 

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. ... 

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. 

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