Increased nucleophile reactivity of amino acid β-naphthylamides in α-chymotrypsin-catalyzed peptide synthesis

Gololobov, Mikhail Yu.; Petrauskas, Alanas; Pauliukonis, Ričardas; Koschke, Vibke; Borisov, Ivan L.; Švedas, Vytas K.

doi:10.1016/0167-4838(90)90124-x

Cited by 10 publications

(16 citation statements)

References 34 publications

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“…Glycoside hydrolases (Adlercreutz, 2017;Seibel et al, 2006;Vera et al, 2020;Zeuner, Jers, Mikkelsen, & Meyer, 2014), lipases (Adlercreutz, 2017;Subileau et al, 2017), amidases (Bruggink, Roos, & de Vroom, 1998;Giordano et al, 2006;Gololobov et al, 1990;Sio & Quax, 2004;Sklyarenko, El'darov, Kurochkina, & Yarotsky, 2015), and phosphatases (Ishikawa et al, 2002;Tasnádi, Staśko, Ditrich, Hall, & Faber, 2020; are industrial enzymes used in these synthetic processes. Mechanistically, the enzymatic reactions proceed in two catalytic steps via a covalent enzyme intermediate (Gololobov, Borisov, Belikov, & Švedas, 1988;Kasche, 1986;Marsden et al, 2020;Seibel et al, 2006;Zeuner et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

“…The first step involves group transfer from the donor substrate to an active-site nucleophile of the enzyme. The enzyme intermediate then reacts with the incoming acceptor (which is water in the canonical hydrolysis reaction) to release the synthetic product (Gololobov et al, 1988(Gololobov et al, , 1990Huber, Kurz, & Wallenfels, 1976;Kasche, 1986;Marsden et al, 2020;Vera et al, 2020;Youshko, Chilov, Shcherbakova, & Švedas, 2002). Synthesis in an aqueous environment operates in competition with hydrolysis (Adlercreutz, 2017;Giordano et al, 2006;Gololobov et al, 1988;Kasche, 1986;Subileau et al, 2017;Wildberger, Pfeiffer, Brecker, Rechberger, et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

“…The natural moisturizer α-D-glucosyl-sn-glycerol (GG), produced industrially using glycosylation of glycerol catalyzed by sucrose phosphorylase (SucP; Bolivar, Luley-Goedl, Leitner, Sawangwan, & Nidetzky, 2017;Goedl, Sawangwan, Mueller, Schwarz, & Nidetzky, 2008;Luley-Goedl, Sawangwan, Mueller, Schwarz, & Nidetzky, 2010), represents a number of hydrolase-promoted transfer reactions for which the reaction scheme shown in Figure 1 is likely an oversimplification (e.g., Fernandez-Lafuente, Rosell, & Guisan, 1998;Gololobov et al, 1988Gololobov et al, , 1990Kasche, 1986;Terreni et al, 2005;Vera, Guerrero, Wilson, & Illanes, 2017;Youshko et al, 2002). The SucP can use sucrose (1) or G1P (2) as donor for GG (3) formation (Goedl, Sawangwan, et al, 2008;Goedl, Schwarz, Minani, & Nidetzky, 2007;Renirie, Pukin, van Lagen, & Franssen, 2010).…”

Section: Introductionmentioning

confidence: 99%

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On the donor substrate dependence of group‐transfer reactions by hydrolytic enzymes: Insight from kinetic analysis of sucrose phosphorylase‐catalyzed transglycosylation

Klimacek

Sigg

Nidetzky

2020

Biotech & Bioengineering

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Chemical group‐transfer reactions by hydrolytic enzymes have considerable importance in biocatalytic synthesis and are exploited broadly in commercial‐scale chemical production. Mechanistically, these reactions have in common the involvement of a covalent enzyme intermediate which is formed upon enzyme reaction with the donor substrate and is subsequently intercepted by a suitable acceptor. Here, we studied the glycosylation of glycerol from sucrose by sucrose phosphorylase (SucP) to clarify a peculiar, yet generally important characteristic of this reaction: partitioning between glycosylation of glycerol and hydrolysis depends on the type and the concentration of the donor substrate used (here: sucrose, α‐d‐glucose 1‐phosphate (G1P)). We develop a kinetic framework to analyze the effect and provide evidence that, when G1P is used as donor substrate, hydrolysis occurs not only from the β‐glucosyl‐enzyme intermediate (E‐Glc), but additionally from a noncovalent complex of E‐Glc and substrate which unlike E‐Glc is unreactive to glycerol. Depending on the relative rates of hydrolysis of free and substrate‐bound E‐Glc, inhibition (Leuconostoc mesenteroides SucP) or apparent activation (Bifidobacterium adolescentis SucP) is observed at high donor substrate concentration. At a G1P concentration that excludes the substrate‐bound E‐Glc, the transfer/hydrolysis ratio changes to a value consistent with reaction exclusively through E‐Glc, independent of the donor substrate used. Collectively, these results give explanation for a kinetic behavior of SucP not previously accounted for, provide essential basis for design and optimization of the synthetic reaction, and establish a theoretical framework for the analysis of kinetically analogous group‐transfer reactions by hydrolytic enzymes.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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On the donor substrate dependence of group‐transfer reactions by hydrolytic enzymes: Insight from kinetic analysis of sucrose phosphorylase‐catalyzed transglycosylation

Klimacek

Sigg

Nidetzky

2020

Biotech & Bioengineering

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“…These complexes form in papain-catalyzed and carboxypeptidase-Y-catalyzed reactions [23, 241. The EN complex is formed in the a-chymotrypsin-catalyzed reactions, if amino acid P-naphthylamides are the added nucleophiles [7]. However, if amino-acid amides are the acceptors in the a-chymotrypsin-catalyzed reactions, neither the EN or ESN complexes form.…”

Section: Do the En And Esn Complexes Form?mentioning

confidence: 99%

“…The dependence of the [PP]l[P,] ratio on the concentration of the amino-acid amides for the a-chymotrypsin-catalyzed transfer of the BzTyr group from BzTyrOEt to ApeNH, ( 0 ) and MetNH, (W). The dependences were interpreted by model C. The lines were drawn according to Eqn(7) using the values of the kinetic constants obtained from presentation of the experimental data according to Eqn( 5 ) shown in insert A. The following values were used: transfer of the BzTyr group to ApeNH, (O), (k4/k3K,Jc = 4.02 X lo--* mM-', [k,/ (k3KJ'n)]c = 1.78 X lo-' mM-,; transfer of the BzTyr group to MetNH, (W), (k,/k,K,), = 3.02 X mM-', [k6/(k3KnK),)], = 0.746 X mM-'.…”

mentioning

confidence: 99%

The second nucleophile molecule binds to the acyl‐enzyme–nucleophile complex in α‐chymotrypsin catalysis

Gololobov

Stepanov

Voyushina

et al. 1993

European Journal of Biochemistry

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a-Chymotrypsin-catalyzed acyl tranfer was studied using three acyl-group donors (Mal-~-Ala-L-Ala-L-PheOMe, Bz-L-TyrOEt and Ac-L-TrpOEt ; Mal, maleyl; Bz, benzoyl ; OMe, methyl ester; OEt, ethyl ester) and a series of amino-acid amides. Most of the reactions studied can be described by the simplest kinetic model without the nucleophile binding to the acyl-enzyme. The a-chymotrypsin-catalyzed transfer of the Mal-L-Ah-L-Ala-L-Phe group to the amides of L-Phe and L-Tyr showed a linear dependence of the partition constant, p , on the nucleophile concentration which can be interpreted by the hydrolysis of the acyl-enzyme-nucleophile complex. The a-chymotrypsincatalyzed transfer of the Bz-L-Tyr and A c -L -T~~ groups to several amino-acid amides showed unusual behavior which can be interpreted by the kinetic model involving formation of a complex of the acyl-enzyme with two nucleophile molecules. These observations can explain the conflicting conclusions concerning the kinetics of a-chymotrypsin-catalyzed acyl transfer evident in previous studies.In proteinase-catalyzed acyl-transfer reactions, the acyl group of a substrate can be transferred both to water and to the added nucleophile. As a result, the hydrolytic product donor, N is the acyl-group acceptor (added nucleophile, amino component), P is the peptide synthesized, P, and P, are the products of the hydrolysis of the acyl-group donor and ES is the enzyme complex with S and EA denoting the acyl-enzyme. The kinetic constants are indicated for the appropriate reactions in the scheme. where E represents the enzyme, S denotes the acyl-group

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Peptide synthesis with a proteinase from the extremely thermophilic organism Thermus Rt41A

Wilson¹,

Daniel

Peek

1994

Biotech & Bioengineering

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A proteinase isolated from Thermus RT41a was immobilized to controlled pore glass beads and was used in the free and immobilized forms for peptide synthesis. The observed maximum yield was the same in both cases. a number of dipeptides were produced from amino acid esters and amides. The best acyl components, from those tested, were found to be Ac-Phe-OEt and Bz-Ala-OMe. Tur-NH(2), Trp-NH(2), Leu-pNA, and Val-pNA were all reactive nucleophiles.The kinetically controlled synthesis of Bz-ala-Tyr-NH(2) was optimized by studying the effect of pH, temperature, solvent concentration, ionic strength, and nucleophile and acyl donor concentration, ionic strength, and nucleophile and acyl donor concentration on the maximum yield. The initial conditions used were 25 mM Bz-ala-OMe, 25 mM Tyr-NH(2), 70 degrees C, pH 8.0, and 10% v/v dimethylformamide (DMF). The optimum conditions were 90% v/v DMF using 80 mM bz-Ala-OMe and 615 mM Tyr-NH(2) at 40 degrees C and pH 10. These conditions increased the maximum conversion from 0.75% to 26% (of the original ester concentration). In a number of other cosolvents, the best peptide yields were observed with acetonitrile and ethyl acetate. In 90% acetonitrile similar yields were observed to those in 90% DMF under optimized conditions except that the acyl donor and nucleophile concentrations could be reduced to 25 mM and 100mM, respectively. The effect of the blocking group on the nucleophile was also investigated; -betaNA and -pNA as blocking groups improved the yields markedly. The blocking and leaving groups of the acyldonor had no effect on the dipeptide yield.

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Increased nucleophile reactivity of amino acid β-naphthylamides in α-chymotrypsin-catalyzed peptide synthesis

Cited by 10 publications

References 34 publications

On the donor substrate dependence of group‐transfer reactions by hydrolytic enzymes: Insight from kinetic analysis of sucrose phosphorylase‐catalyzed transglycosylation

On the donor substrate dependence of group‐transfer reactions by hydrolytic enzymes: Insight from kinetic analysis of sucrose phosphorylase‐catalyzed transglycosylation

The second nucleophile molecule binds to the acyl‐enzyme–nucleophile complex in α‐chymotrypsin catalysis

Peptide synthesis with a proteinase from the extremely thermophilic organism Thermus Rt41A

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