Chemical synthesis and papain-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-lysine p-nitroanilide.
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ABSTRACT:
SUBMITTER: Mackenzie NE
PROVIDER: S-EPMC1144749 | biostudies-other | 1985 Mar
REPOSITORIES: biostudies-other
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Project description:1. The Michaelis-Menten parameters for the papain-catalysed hydrolysis of a number of alkyl, aryl and alkyl-thiol esters of hippuric acid have been determined. 2. For all the aryl esters and most of the alkyl esters studied, the catalytic constant, k(0), is 2-3sec.(-1) and most probably represents deacylation of the common intermediate, hippuryl-papain. 3. Two alkyl esters and hippurylamide, however, have catalytic rate constants, k(0), less than 2-3sec.(-1). It is possible to interpret all the available kinetic data in terms of a three-step mechanism in which an enzyme-substrate complex is first formed, followed by acylation of the enzyme through an essential thiol group, followed by deacylation of the acyl-enzyme. 4. The logarithm of the ratio of the Michaelis-Menten parameters, which reflect the acylation rate constant, for four aryl esters of hippuric acid studied give a linear Hammett plot against the substituent constant, sigma. Arguments are presented that indicate acid as well as nucleophilic catalysis in the acylation process and that the most likely proton donor is an imidazolium ion. 5. It is suggested that this imidazolium ion is part of the same histidine residue that has been tentatively implicated in the deacylation process (Lowe & Williams, 1965b). 6. A new mechanism is proposed for the papain-catalysed hydrolysis of N-acyl-alpha-amino acid derivatives.
| S-EPMC1206922 | biostudies-other
Project description:A detailed study of the kinetics of the trypsin (EC 3.4.21.4)-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-lysine p-nitrophenyl ester in cryosolvents at 0 degrees C and below was undertaken. The pH-dependences of kcat, Km, k+2, k+3 and Ks were determined under cryoenzymological conditions and are compared with previous results [Antonini & Ascenzi (1981) J. Biol. Chem. 256, 12449-12455] obtained in fully aqueous media at ambient temperatures. Below pH 5.0 the kinetics, and presumably the mechanism of catalysis, are not significantly perturbed under cryoenzymological conditions. However, it is shown that below pH 5.0 both Km and Ks are decreased under these conditions but that both are increased at pH 6.7 relative to the results obtained in fully aqueous media at ambient temperatures. The effects of the cryoenzymological conditions on the individual catalytic parameters are discussed. The acylation rate constant, k+2, is essentially constant at pH 4.2 and 5.0 but decreases at lower pH values with an apparent pKa of approx. 4.0. In view of the low enthalpy of ionization associated with this pKa it is suggested that this group is the carboxy group of aspartic acid-189, which binds the positively charged lysine side chain of the substrate. The mechanistic implications of the results for the acylation step are discussed. It is also shown that only at low pH values can significant amounts of acylated trypsin be accumulated.
| S-EPMC1152436 | biostudies-other
Project description:This study shows for the first time that boric acid catalyses the hydrolysis of peroxyacids, resulting in an approximately 12-fold increase in hydrolysis rate for both peracetic acid (PAA) and 3-chloroperbenzoic acid (MCPBA) when 0.1 M boric acid is present. The maximum rate of hydrolysis occurs at pH 9 and pH 8.4 for PAA and MCPBA respectively. In contrast, carbonate buffer does not enhance the rate of PAA hydrolysis. The reaction was followed by measuring the initial rate of hydrogen peroxide formation using a specific Ti(iv) complexation method. The study of the hydrolysis reaction requires the presence of 2 × 10-5 M each of ethylenediaminetetraacetic acid (EDTA) and ethylenediamine tetramethylene phosphonic acid (EDTMP) in all solutions in order to chelate metal ions across the full pH range (3 to 13) that would otherwise contribute to peroxyacid decomposition. Catalysis of peroxyacid hydrolysis is most likely effected by the triganol boric acid acting as a Lewis acid catalyst, associating with the peroxide leaving group in the transition state to reduce the leaving group basicity. The products of the reaction are the well characterised monoperoxoborate species and the parent carboxylic acid. Analysis of the pH and borate dependence data reveals that in addition to a catalytic pathway involving a single boric acid molecule, there is a significant pathway involving either (a) two boric acid molecules or (b) the polyborate species, B3O3(OH)4 -. Knowledge about catalytic mechanisms for the loss of peroxyacids through hydrolysis is important because they are widely used in reagents in a range of oxidation, bleaching and disinfection applications.
| S-EPMC10103745 | biostudies-literature
Project description:The pH-dependence of the Michaelis-Menten parameters for the papain-catalysed hydrolysis of N-acetyl-l-phenylalanylglycine p-nitroanilide was determined. The equilibrium binding constant, K(s), is independent of pH between 3.7 and 9.3, whereas the acylation constant, k(+2), shows bell-shaped pH-dependence with apparent pK(a) values of 4.2 and 8.2. The effect of substituents in the leaving group on the acylation constant of the papain-catalysed hydrolysis of hippuryl anilides and N-acetyl-l-phenylalanylglycine anilides gives rise in both series to a Hammett rho value of -1.04. This indicates that the enzyme provides electrophilic, probably general-acid, catalysis, as well as the nucleophilic or general-base catalysis previously found. A mechanism involving a tetrahedral intermediate whose formation is general-base-catalysed and whose breakdown is general-acid-catalysed seems most likely. The similarity of the Hammett rho values appears to exclude facilitated proton transfer as a means through which the specificity of papain is expressed.
| S-EPMC1177120 | biostudies-other
Project description:Closed-loop recycling offers the opportunity to mitigate plastic waste through reversible polymer construction and deconstruction. Although examples of chemical recycling of polymers are known, few have been applied to materials derived from abundant commodity olefinic monomers, which are the building blocks of ubiquitous plastic resins. Here we describe a [2+2] cycloaddition/oligomerization of 1,3-butadiene to yield a previously unrealized telechelic microstructure of (1,n'-divinyl)oligocyclobutane. This material is thermally stable, has stereoregular segments arising from chain-end control, and exhibits high crystallinity even at low molecular weight. Exposure of the oligocyclobutane to vacuum in the presence of the pyridine(diimine) iron precatalyst used to synthesize it resulted in deoligomerization to generate pristine butadiene, demonstrating a rare example of closed-loop chemical recycling of an oligomeric material derived from a commodity hydrocarbon feedstock.
| S-EPMC7875253 | biostudies-literature
Project description:The Sir2 family of enzymes or sirtuins are known as nicotinamide adenine dinucleotide (NAD)-dependent deacetylases and have been implicated in the regulation of transcription, genome stability, metabolism and lifespan. However, four of the seven mammalian sirtuins have very weak deacetylase activity in vitro. Here we show that human SIRT6 efficiently removes long-chain fatty acyl groups, such as myristoyl, from lysine residues. The crystal structure of SIRT6 reveals a large hydrophobic pocket that can accommodate long-chain fatty acyl groups. We demonstrate further that SIRT6 promotes the secretion of tumour necrosis factor-α (TNF-α) by removing the fatty acyl modification on K19 and K20 of TNF-α. Protein lysine fatty acylation has been known to occur in mammalian cells, but the function and regulatory mechanisms of this modification were unknown. Our data indicate that protein lysine fatty acylation is a novel mechanism that regulates protein secretion. The discovery of SIRT6 as an enzyme that controls protein lysine fatty acylation provides new opportunities to investigate the physiological function of a protein post-translational modification that has been little studied until now.
| S-EPMC3635073 | biostudies-literature
Project description:The proteinase-catalysed synthesis of [Leu]enkephalin and [Met]enkephalin was studied kinetically. N alpha-t-Butoxycarbonyl-amino acids and peptides or their ethyl esters served as acyl donors, and amino acid phenylhydrazides were used as acyl acceptors. Initial-velocity measurements of alpha-chymotrypsin-catalysed peptide synthesis gave rise to kinetic patterns that are compatible with a ping-pong mechanism modified by a hydrolytic branch. Initial-rate and alternative-substrate inhibition patterns for papain-controlled peptide-bond formation are consistent with a sequential ordered mechanism with the acyl donor as the obligatory first substrate. On the basis of the observed kinetic features, reaction mechanisms are proposed for chymotrypsin- and papain-catalysed peptide synthesis that inversely equal those describing the pathways of proteolysis. The respective initial-velocity expressions for bireactant systems are given, along with the numerical values of the corresponding kinetic parameters.
| S-EPMC1153641 | biostudies-other
Project description:The hydrophobic central cavity of a water-soluble M8 L12 cubic coordination cage can accommodate a range of phospho-diester and phospho-triester guests such as the insecticide "dichlorvos" (2,2-dichlorovinyl dimethyl phosphate) and the chemical warfare agent analogue di(isopropyl) chlorophosphate. The accumulation of hydroxide ions around the cationic cage surface due to ion-pairing in solution generates a high local pH around the cage, resulting in catalysed hydrolysis of the phospho-triester guests. A series of control experiments unexpectedly demonstrates that-in marked contrast to previous cases-it is not necessary for the phospho-triester substrates to be bound inside the cavity for catalysed hydrolysis to occur. This suggests that catalysis can occur on the exterior surface of the cage as well as the interior surface, with the exterior-binding catalysis pathway dominating here because of the small binding constants for these phospho-triester substrates in the cage cavity. These observations suggest that cationic but hydrophobic surfaces could act as quite general catalysts in water by bringing substrates into contact with the surface (via the hydrophobic effect) where there is also a high local concentration of anions (due to ion pairing/electrostatic effects).
| S-EPMC7079011 | biostudies-literature
Project description:The specificity of the proteolytic enzyme, papain, for the peptide bond of the substrate adjacent to that about to be cleaved and for the acyl residue of some N-acylglycine derivatives is manifest almost exclusively in the formation of the acyl-enzyme from the enzyme-substrate complex. Models for the enzyme-substrate complex and acyl-enzyme intermediate are suggested that account for these observations. In particular it is suggested that the peptide bond of the substrate adjacent to that about to be cleaved, is bound in the cleft of the enzyme between the NH group of glycine-66 and the backbone C=O group of aspartic acid-158, and provides a sensitive amplification mechanism through which the specificity of the enzyme for hydrophobic amino acids such as l-phenylalanine is relayed. It is also suggested that the distortion in the enzyme-substrate complex and the binding of the peptide bond adjacent to that about to be cleaved are also linked and behave co-operatively, the distortion of the protein facilitating binding and the stronger binding facilitating distortion. The results imply that between the enzyme-substrate complex and the acyl-enzyme a relaxation of the protein conformation must occur.
| S-EPMC1177119 | biostudies-other
Project description:A copper-catalysed regio- and diastereoselective borylamination of α,β-unsaturated esters with B2pin2 and hydroxylamines has been developed to deliver acyclic β-boryl-α-amino acid derivatives with high anti-diastereoselectivity (up to >99 : 1), which is difficult to obtain by the established methods. A chiral phosphoramidite ligand also successfully induces the enantioselectivity, giving the optically active β-borylated α-amino acids. The products can be stereospecifically transformed into β-functionalised α-amino acids, which are of potent interest in medicinal chemistry.
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