Dimiter D. Petkov

Enzymic Synthesis Design and Enzymic Synthesis of Aspartame

Abstract An enzymic synthesis of aspartame (H-Asp-Phe-OMe) has been designed and realized based on the structure-activity study of thermolysin and penicillin amidase hydrolysis of its p-substituted phenylacetyl derivatives. These compounds meet the structural and energetic requirements of two enzymic binding sites The peptide sweetener has been prepared by thermolysin - catalyzed condensation of the p-substituted phenylacetyl-Asp-OH and H-Phe-OMe followed by penicillin amidase - catalyzed deprotection of the resulted aspartame precursors.

Enzyme peptide synthesis by an iterative procedure in a nucleophile pool

Abstract An effective method for enzyme solubility-controlled synthesis of peptides, consisting in an iterative addition of equivalent amounts of acyl and amine components to a solution (nucleophile pool) containing the enzyme and a large excess of the amine component, is described.

β-Aspartylpeptides as substrates of L-asparaginases from Escherichia coli and Erwinia chrysanthemi

L‐Asparaginase is known to catalyze the hydrolysis of L‐asparagine to L‐aspartic and ammonia, but little is known about its action on peptides. When we incubated L‐asparaginases purified either from Escherichia coli or Erwinia chrysanthemi – commonly used as chemotherapeutic agents because of their antitumour activity – with eight small β‐aspartylpeptides such as β‐aspartylserineamide, β‐aspartylalanineamide, β‐aspartylglycineamide and β‐aspartylglycine, we found that both L‐asparaginases could catalyze the hydrolysis of five of them yielding L‐aspartic acid and amino acids or peptides. Our data show that L‐asparaginases can hydrolyze β‐aspartylpeptides and suggest that L‐asparaginase therapy may affect the metabolism of β‐aspartylpeptides present in human body.

Glycosylasparaginase-catalyzed synthesis and hydrolysis of beta-aspartyl peptides.

β-Aspartyl di- and tripeptides are common constituents of mammalian metabolism, but their formation and catabolism are not fully understood. In this study we provide evidence that glycosylasparaginase (aspartylglucosaminidase), an N-terminal nucleophile hydrolase involved in the hydrolysis of the N-glycosidic bond in glycoproteins, catalyzes the hydrolysis of β-aspartyl peptides to form l-aspartic acid and amino acids or peptides. The enzyme also effectively catalyzes the synthesis of β-aspartyl peptides by transferring the β-aspartyl moiety from other β-aspartyl peptides or β-aspartylglycosylamine to a variety of amino acids and peptides. Furthermore, the enzyme can usel-asparagine as the β-aspartyl donor in the formation of β-aspartyl peptides. The data show that synthesis and degradation of β-aspartyl peptides are new, significant functions of glycosylasparaginase and suggest that the enzyme could have an important role in the metabolism of β-aspartyl peptides.

Recombinant human glycosylasparaginase catalyzes hydrolysis of L-asparagine

Glycosylasparaginase is a lysosomal amidase involved in the degradation of glycoproteins. Recombinant human glycosylasparaginase is capable of catalyzing the hydrolysis of the amino acid l‐asparagine to l‐aspartic acid and ammonia. For the hydrolysis of l‐asparagine the K m is 3–4‐fold higher and V max 1/5 of that for glycoasparagines suggesting that the full catalytic potential of glycosylasparaginase is not used in the hydrolysis of the free amino acid. l‐Asparagine competitively inhibits the hydrolysis of aspartylglucosamine indicating that both the amino acid and glycoasparagine are interacting with the same active site of the enzyme. The hydrolytic mechanism of l‐asparagine and glycoasparagines will be discussed.

N-anthraniloylation converts peptide p-nitroanilides into fluorogenic substrates of proteases without loss of their chromogenic properties

By simple substitution of an N-acyl group for the anthraniloyl(o-aminobenzoyl) group, chromogenic p-nitroanilide substrates are converted into highly sensitive fluorogenic substrates of proteases. The fluorescence of the anthraniloyl group is completely quenched by the p-nitroanilide moiety in the intact substrates and is released during their enzymatic hydrolysis. The approach is exemplified by the synthesis of anthraniloyl-Phe p-nitroanilide, anthraniloyl-Lys p-nitroanilide, and anthraniloyl-Gly-Gly-Phe p-nitroanilide as substrates for chymotrypsin, trypsin, and alkaline mesentericopeptidase, respectively. The kinetic parameters of these substrates are slightly better than those of similar derivatives bearing other acyl groups, suggesting that the enhanced sensitivity is completely due to the method of measurement. Since the conversion does not affect the chromogenic properties of the substrates, the same compounds can be used as usual p-nitroanilide substrates as well.

Phenylacetyl group as enzyme-cleavable aminoprotection of purine nucleosides

Abstract N 6 -Phenylacetyl-2′-deoxyadenosine and N 2 -Phenylacetyl-2′-deoxyguanosine are readily deprotected in reactions catalyzed by free and immobilized penicillin amidase at pH 7.8 and 25°C.

Chemical-enzymatic incorporation of D-amino acids into peptides: synthesis of diastereomeric (D-Ala2, D-Leu5)enkephalinamides

Enzymatic synthesis Opioid peptide Enkephalin NMR

Catalysis and leaving group binding in anilide hydrolysis by chymotrypsin

The influence of the leaving group on the reactivity of specific anilides in alpha-chymotrypsin-catalyzed hydrolysis (chymotrypsin, EC 3.4.21.2) involves both its binding to the enzyme (steric effect) and electronic nature (electronic effect). These effects are considered in terms of the stereoelectronic theory for the formation and cleavage of the tetrahedral intermediate in acyltransfer reactions. The application of this theory to the enzyme hydrolysis leads to the conclusion that the nature of the reaction products and the effectiveness of the catalysis are controlled by the orientation of the leaving group nitrogen lone pair orbital. The leaving group binding affects the formation of a reactive conformation of the enzyme tetrahedral intermediate that is presumed to intervene between the Michaelis complex and the acylenzyme. The steric and electronic effects could be separated in a straightforward fashion only in the case of equal binding of the leaving groups to the leaving-group-binding site of alpha-chymotrypsin.

Yield optimization in the kinetically controlled enzymic peptide synthesis

Abstract The yield and its time-dependence in acylenzyme mechanism-based enzymic peptide synthesis are controlled by the proteinase kinetic specificity. The maximum yield is limited by a non-equilibrium constant K max . Both K max and the time, t max , taken to attain the maximum yield, are directly related to the enzyme kinetic parameters. These relationships allow kinetic determination of yield optimization in kinetically controlled enzymic peptide synthesis.