The thermolysin-catalyzed condensation reactions of n-substituted aspartic and glutamic acids with phenylalanine alkyl esters

Isowa, Yoshikazu; Ohmori, Muneki; Ichikawa, Tetsuya; Mori, Katsuharu; Nonaka, Yasuki; Kihara, Keiichi; Oyama, Kiyotaka; SATOH, H.; Nishimura, Shigeaki

doi:10.1016/s0040-4039(01)86363-2

Cited by 112 publications

(32 citation statements)

References 17 publications

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“…3 might be ascribable to the Z-Asp-PheOMe produced during the reaction forming an insoluble complex with PheOMe and precipitating in the reaction mixture [4]. Eqn (4) is further rearranged with the equations for dissociation of Z-Asp, PheOMe and Z-AspPheOMe (Eqns 5 -8), giving Eqn (9).…”

Section: Dependence On Ph Of the Equilibrium Yield Of Z-asp-pheome Inmentioning

confidence: 99%

Kinetics and equilibrium of enzymatic synthesis of peptides in aqueous/organic biphasic systems

Nakanishi

Kimura

Matsuno

1986

European Journal of Biochemistry

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We studied kinetics and the equilibrium relationship for the thermolysin-catalyzed synthesis of N-(benzyloxycarbony1)-L-aspartyl-L-phenylalanine methyl ester (Z-Asp-PheOMe) from N-(benzyloxycarbony1)-L-aspartic acid (Z-Asp) and L-phenylalanine methyl ester (PheOMe) in an aqueous-organic biphasic system. This is a model reaction giving a condensation product with dissociating groups. The kinetics for the synthesis of Z-Asp-PheOMe in aqueous solution saturated with ethyl acetate was expressed by a rate equation for the rapid-equilibrium random bireactant mechanism, and the reverse hydrolysis reaction was zero-order with respect to Z-Asp-PheOMe concentration. The courses of synthesis of Z-Asp-PheOMe in the biphasic system were well explained, by the rate equations obtained for the aqueous solution and by the partition of substrate and condensation product between the both phases. The rate of synthesis in the biphasic system was much lower than in aqueous solution due to the unfavorable partition of PheOMe in the aqueous phase.The equation for the equilibrium yield of Z-Asp-PheOMe in the biphasic system was derived assuming that only the non-ionized forms of the substrate and condensation product exist in the organic phase. It was found theoretically and experimentally that the yield of Z-Asp-PheOMe is maximum at the aqueous-phase pH of around 5, lower than for synthesis in aqueous solution. The effect of the organic solvent on the rate and equilibrium for the synthesis of Z-Asp-PheOMe could be explained by the variation in the partition coefficient.The effect of the partitioning of substrate on the aqueous-phase pH change was also shown.The kmetics of the thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-phenylalanine methyl ester (Z-PhezOMe) in an aqueous/organic biphasic system has been discussed in the preceding paper [l]. The condensation product, Z-Phe20Me is highly insoluble in aqueous solution, and it is found almost exclusively in the organic solvent in a biphasic system, so the reaction can be analyzed as being almost irreversible. Here, we studied the thermolysin-catalyzed synthesis of N-(benzyloxycarbony1)-L-aspartyl-L-phenylalanine methyl ester (Z-Asp-PheOMe), a precursor of the synthetic sweetener aspartame [2], by condensing N-(benzyloxycarbony1)-L-aspartic acid (Z-Asp) and L-phenylalanine methyl ester (PheOMe), as a model reaction forming a soluble condensation product. The overall reaction mechanism is expressed by Eqn (1).Z-Asp-PheOMe is more soluble (about 40 mM at 40°C and pH 6) than Z-PhezOMe (about 5 pM at 40°C and pH 7) be- cause of its side carboxylic acid. The purpose of this study was to elucidate theoretically and experimentally the equilibrium yield of the reaction as well as the rate of the synthesis of Z-Asp-PheOMe in an aqueous/organic biphasic system. Various factors affecting the equilibrium yield and reaction rate, such as the aqueous-phase pH, the volume ratio of the organic to aqueous phases, and the organic solvent, were examined quantitatively.

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Section: Dependence On Ph Of the Equilibrium Yield Of Z-asp-pheome Inmentioning

confidence: 99%

Kinetics and equilibrium of enzymatic synthesis of peptides in aqueous/organic biphasic systems

Nakanishi

Kimura

Matsuno

1986

European Journal of Biochemistry

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“…This is related to the fact that the peptide condensation reaction by this enzyme is largely equilibrium-controlled for a combination of specific substrates. 2 It is noted that the modified lm-TLN has catalytic activity for the condensation reaction of specific substrates comparable to the native one and the effect of modification is only an increase in activity for the condensation of less specific substrates, i.e., to broaden substrate specificity.…”

Section: Resultsmentioning

confidence: 98%

Effect of Acylation on Peptide Synthesis by Immobilized Thermolysin

Kunugi

Morikawa

Kondoh

et al. 1988

Polym J

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KEY WORDSImmobilized Enzyme / Thermolysin / Acylation / Chemical Modification / Peptide Synthesis / A natural enzyme often shows strict specificity so that it is hard to use for a reaction of less specific substrates. Modulation of the enzyme function through structure, by either genetic or chemical procedure may eliminate this difficulty. A number of studies on chemical modification of enzyme has been performed to elucidate the detailed reaction mechanism and the results are very interesting for practical use of enzyme reactions. One example is the peptide formation catalyzed by a proteolytic enzyme. Here we take thermolysin, a thermostable neutral protease from B. thermoproteoliticus, 1 as the target. This enzyme can catalyze a condensation of N-acylamino acid and amino acid amide (eq 1: AC, acylgroup; AA and AA', amino acid residue). 2 AC-AA+AA'-NH 2 ~AC-AA-AA'-NH2 (1) We have investigated several kinetic aspects of free and immobilized thermolysin. 3 • 4 On the mechanism of this enzyme Vallee's school reported extensive studies 5 -7 and showed that this enzyme exhibits different catalytic behavior when the active site zinc ion is replaced by cobalt ion 7 or when Tyr-110 was acylated with N-acetylamino acid. 6 The latter result * To whom all correspondence should be addressed.was further certified by another independent group. 8 Though a peptide condensation reaction is the reverse reaction of hydrolysis, there are many factors controlling the rate and yield and therefore the effect of chemical modification on the condensation reaction cannot be estimated straightforward from the results of hydrolytic reactions.Recently, studies on the effect of metal substitution of thermolysin on the peptide synthesis were reported for the free (soluble) and immobilized states of an enzyme. 9 • 10 Here we examine the effect of acylation on a few simple condensation reactions catalyzed by thermolysin immobilized on glass matrices. The immobilized state is chosen for ease of various chemical treatments and feasibility of applications. Since immobilization of an enzyme is already a "chemical" modification, this example is a sort of double modification.

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“…In 1979, Isowa et al 60 showed that the chiral aspartame precursor Z-L-Asp-L-Phe-OMe could be produced from the thermolysin-promoted condensation of Z-L-Asp and L-Phe-OMe. The results were confirmed by Wayne and Fruton 61 and Nakanishi et al 62,63 The rate of reaction is, however, slow.…”

Section: U S E O F T H E R M O L Y S I N I N P E P T I D E C O U P L mentioning

confidence: 99%

Thermolysin

Matthews

2004

Handbook of Metalloproteins

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Enzyme: thermolysin (TLN) (E.C.3.4.24.4); a metalloprotease with a single zinc ion in the active site. The enzyme also binds four calcium ions that contribute to thermostability.Thermolysin is an endopeptidase of molecular weight 34 600 Da isolated from the thermophile Bacillus thermoproteolyticus.1 It preferentially cleaves peptides with a bulky, hydrophobic residue (phenylalanine or leucine) in the R 1 position. [2][3][4] As noted below, thermolysin is a member of a very large family and the different family members display a broad range of specificities. O C C U R R E N C EThermolysin is the prototypical member of the socalled 'M4' or 'thermolysin' family of secreted eubacterial endopeptidases defined by Rawlings and Barrett. 5 All members of the family contain the HEXXH sequence motif in which the two histidines coordinate the zinc ion and the 3D Structure Ribbon drawing showing the overall structure of thermolysin. The view is toward the active site with the zinc ion shown in light blue. A bound inhibitor (ZF P LA) is shown in dark blue. Four calcium ions are shown as green spheres. The amino-terminal domain is located in the top half of the figure. It includes an extended β-sheet that terminates across the 'top' of the substrate binding site and contributes to substrate binding. The carboxy-terminal domain, located in the bottom half of the figure, is predominantly α-helical.[ Figure, based Thermolysin glutamic acid is critical for catalysis (see below). Family members include enzymes from the pathogens Legionella, Listeria, Pseudomonas, and Vibrio. 5 Proteins that are structurally and functionally related to thermolysin, and presumably evolved from a common precursor, extend well beyond the M4 family. Rawlings and Barrett 5 define the 'HEXXH + E Metallopeptidase Clan (MA).' This includes families M4 (thermolysin), M5 (mycolysin), M13 (neprilysin), M1 (membrane alanyl aminopeptidase), and M2 (peptidyl-dipeptidase A). All members of these families are clearly related to thermolysin. Rawlings and Barrett 5 also define the 'HEXXH + H Metallopeptidase Clan (MB).' This includes families M12 (astacin and reprolysin), M10 (interstitial collagenase, serralysin, and matrixins), M11 (autolysin), and M7 (Streptomyces small neutral protease). Members of the latter clan have also been defined as the metzincins 6 and have been shown to have significant structural relationship to thermolysin. B I O L O G I C A L F U N C T I O NThermolysin and its immediate family members are secreted eubacterial peptidases. As such, the presumed biological function of the enzyme is to degrade ambient peptides that can be utilized by the host. More distantly related relatives are involved in a broad range of digestive activities. They also are involved in the processing of biologically active peptides. Perhaps the best-known example is that of the angiotensin-converting enzyme for which the structure has recently been determined. 7 A M I N O A C I D S E Q U E N C E I N F O R M A T I O NThe amino acid sequence of thermolysin was deter...

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The thermolysin-catalyzed condensation reactions of n-substituted aspartic and glutamic acids with phenylalanine alkyl esters

Cited by 112 publications

References 17 publications

Kinetics and equilibrium of enzymatic synthesis of peptides in aqueous/organic biphasic systems

Kinetics and equilibrium of enzymatic synthesis of peptides in aqueous/organic biphasic systems

Effect of Acylation on Peptide Synthesis by Immobilized Thermolysin

Thermolysin

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