Hydrolysis Of Specific Substrates Research Articles

Bioinspired design of highly specific fluorescent probe for butyrylcholinesterase imaging in living cells and Alzheimer's disease model

Butyrylcholinesterase (BChE) is associated with many neurodegenerative diseases, especially Alzheimer’s disease (AD). Developing specific imaging tools for BChE is now highly pursued because BChE has substantially similar biochemical properties to acetylcholinesterase (AChE) but distinct roles in AD. Inspired by the BChE-catalyzed hydrolysis of specific substrates (benzoylcholine, tetracaine, and cocaine) containing benzoyl group in physiological condition, a bioinspired probe PM1 with high specificity towards BChE was designed. The docking models indicated that PM1 could easily access the active center of BChE rather than AChE, showing the significance of benzoyl group in specific recognition of BChE. The control probes PM2–5 with smaller recognition groups displayed lower specificity to BChE than PM1, showing the high efficiency of the bioinspired design strategy. The living cells experiments showed that PM1 signals were weakened by BChE inhibitor but not influenced by AChE inhibitor, which was in contrast to the performance of probe PM2–5, showing the specific recognition of PM1 for BChE in biological system. Moreover, PM1 has been used to study the positive correlation between BChE level and cell apoptosis for the first time, and the further study showed that Aβ deposition could induce the increase in BChE level and accompany the occurrence of cell apoptosis. Notably, PM1 was successfully traced the enhancement of BChE activity in the brain slice of AD mouse model. Such a rational approach may provide a solid basis for designing specific probes with different properties to study BChE in biosystems.

Structure-guided engineering of the substrate specificity of a fungal β-glucuronidase toward triterpenoid saponins

Glycoside hydrolases (GHs) have attracted special attention in research aimed at modifying natural products by partial removal of sugar moieties to manipulate their solubility and efficacy. However, these modifications are challenging to control because the low substrate specificity of most GHs often generates undesired by-products. We previously identified a GH2-type fungal β-glucuronidase from Aspergillus oryzae (PGUS) exhibiting promiscuous substrate specificity in hydrolysis of triterpenoid saponins. Here, we present the PGUS structure, representing the first structure of a fungal β-glucuronidase, and that of an inactive PGUS mutant in complex with the native substrate glycyrrhetic acid 3-O-mono-β-glucuronide (GAMG). PGUS displayed a homotetramer structure with each monomer comprising three distinct domains: a sugar-binding, an immunoglobulin-like β-sandwich, and a TIM barrel domain. Two catalytic residues, Glu414 and Glu505, acted as acid/base and nucleophile, respectively. Structural and mutational analyses indicated that the GAMG glycan moiety is recognized by polar interactions with nine residues (Asp162, His332, Asp414, Tyr469, Tyr473, Asp505, Arg563, Asn567, and Lys569) and that the aglycone moiety is recognized by aromatic stacking and by a π interaction with the four aromatic residues Tyr469, Phe470, Trp472, and Tyr473 Finally, structure-guided mutagenesis to precisely manipulate PGUS substrate specificity in the biotransformation of glycyrrhizin into GAMG revealed that two amino acids, Ala365 and Arg563, are critical for substrate specificity. Moreover, we obtained several mutants with dramatically improved GAMG yield (>95%). Structural analysis suggested that modulating the interaction of β-glucuronidase simultaneously toward glycan and aglycone moieties is critical for tuning its substrate specificity toward triterpenoid saponins.

Erratum to: “Self-assembling catalytic systems based on new amphiphile containing purine fragment, exhibiting substrate specificity in hydrolysis of phosphorus acids esters”

Micellar catalytic systems based on new purine-containing amphiphile for hydrolytic decomposition of organophosphorus toxicants have been prepared. The aggregation threshold of the system has been estimated by means of tensiometry, conductometry, spectrophotometry, and dynamic light scattering. The solubilizing action of the amphiphile towards the hydrophobic dye has been demonstrated. Kinetic studies of hydrolysis of phosphonates differing in the hydrophobicity have disclosed the substrate specificity of the micellar catalysts; inhibition instead of catalysis has been observed in the case of the less hydrophobic substrate.

Self-assembling catalytic systems based on new amphiphile containing purine fragment, exhibiting substrate specificity in hydrolysis of phosphorus acids esters

Self-assembling catalytic systems based on new amphiphile containing purine fragment, exhibiting substrate specificity in hydrolysis of phosphorus acids esters

Substrate specificity in hydrolysis and transglucosylation by family 1 β-glucosidases from cassava and Thai rosewood

Abstract Thai rosewood (Dalbergia cochinchinensis Pierre) dalcochinase and cassava (Manihot esculenta Crantz) linamarase are glycoside hydrolase family 1 β-glucosidases with 47% amino acid sequence identity. Each enzyme can hydrolyze its natural substrate, dalcochinin-8′-O-β- d -glucoside and linamarin, respectively, but not the natural substrate of the other enzyme. Linamarase can transfer glucose to primary, secondary and tertiary alcohols with high efficiency, while dalcochinase can transglucosylate primary and secondary alcohols at moderate levels. In this study, eight amino acid residues in the aglycone binding pocket of dalcochinase were individually replaced with the corresponding residues of linamarase, in order to identify residues that may account for their catalytic differences. The residues I185 and V255 of dalcochinase appeared important for its substrate specificity, with their respective mutations resulting in 24- and 12-fold reductions in kcat/Km for the hydrolysis of dalcochinin-8′-O-β- d -glucoside. Transglucosylation activity was improved when I185, N189 and V255 of dalcochinase were replaced with A201, F205 and F271 of linamarase, respectively, suggesting these residues support transglucosylation in linamarase. Among these three mutants, only the N189F mutant showed significant increases in the rate constants for the reactivation of trapped glucosyl-enzyme intermediates by all alcohols. Together, our results suggest that both hydrophobicity and geometry are important determinants for substrate specificity in hydrolysis and transglucosylation by these family 1 β-glucosidases.

Expression of gelatinases A and B and their endogenous regulators in immortal and transformed fibroblasts

Matrix metalloproteinases (MMP) play a critical role in tumor invasion and metastasis. The aim of this study was to elucidate peculiarity of expression of gelatinases A and B (MMP-2 and MMP-9), membrane type MMP (MT1-MMP) and tissue inhibitor of MMP (TIMP-2) in immortal (IF) and transformed fibroblasts (TF).The study was carried out using embryo rat fibroblasts, sequentially immortalized with the polyomavirus LT gene and transformed with the E7 gene of human papilloma virus (HPV-16). Papilloma virus type 16 and 18 are etiological factors of cervical cancer. The primary fibroblast (PF) culture of Fisher rats was used as control. Analysis of TF and IF involved: determination of MMP-2 and MMP-9 activity by hydrolysis of specific substrate--radioactive collagen type IV; obtaining of MMP spectra by zimographic assay and estimation of the mRNA expression (by RT-PCR) of MMP-2, MMP-9, MT1- MMP and TIMP-2. It was found: 1) collagenolytic activity of MMP was increased only in TF and was dependent on the degree of cell tumorogenity; 2) the study of MMP spectra was shown that MMP-9 was found in TF only but MMP-2 was found in all investigated clones; 3) The mRNA expression of MMP-9, MT1-MMP and TIMP-2 was increased in all TF while the MMP-2 expression was increased in TF only after TF cell selection on rats; 4) The collagenolytic activity as well as the mRNA expression of MMP-2 and MMP-9 themselves and of MMP-2 endogenous regulators (MT1-MMP and TIMP-2) did not change in immortalized fibroblasts compared to PF. The data obtained indicate changes in the enzyme/inhibitor/activator ratio and also suggest of a significant increase in the TF destructive potential. MMP-9 is supposed to be a marker of fibroblasts transformed by E7 HPV-16 gene in cell culture.

Isolation and properties of fungal β-glucosidases

Using chromatography on different matrixes, three beta-glucosidases (120, 116, and 70 kDa) were isolated from enzymatic complexes of the mycelial fungi Aspergillus japonicus, Penicillium verruculosum, and Trichoderma reesei, respectively. The enzymes were identified by MALDI-TOF mass-spectrometry. Substrate specificity, kinetic parameters for hydrolysis of specific substrates, ability to catalyze the transglucosidation reaction, dependence of the enzymatic activity on pH and temperature, stability of the enzymes at different temperatures, adsorption ability on insoluble cellulose, and the influence of glucose on catalytic properties of the enzymes were investigated. According to the substrate specificity, the enzymes were shown to belong to two groups: i) beta-glucosidase of A. japonicus exhibiting high specific activity to the low molecular weight substrates cellobiose and pNPG (the specific activity towards cellobiose was higher than towards pNPG) and low activity towards polysaccharide substrates (beta-glucan from barley and laminarin); ii) beta-glucosidases from P. verruculosum and T. reesei exhibiting relatively high activity to polysaccharide substrates and lower activity to low molecular weight substrates (activity to cellobiose was lower than to pNPG).

Cloning and characterization of a novel α-galactosidase fromBifidobacterium breve203 capable of synthesizing Gal-α-1,4 linkage

A novel alpha-galactosidase gene (aga2) was cloned from Bifidobacterium breve 203. It contained an ORF of 2226-bp nucleotides encoding 741 amino acids with a calculated molecular mass of 81.5 kDa. The recombinant enzyme Aga2 was heterogeneously expressed, purified and characterized. Regarding substrate specificity for hydrolysis, Aga2 was highly active towards p-nitrophenyl-alpha-d-galactopyranoside (pNPG). The Km value for pNPG was estimated to be 0.27 mM and for melibiose it was estimated to be 4.3 mM. Aga2 was capable of catalyzing transglycosylation as well as hydrolysis. The enzyme synthesized a trisaccharide (Gal-alpha-1, 4-Gal-alpha-1, 6-Glc) using melibiose as a substrate. It was a new oligosaccharide produced by glycosidase and contained Gal-alpha-1,4 linkage, a novel galactosidic link formed by microbial alpha-galactosidase. In the presence of pNPG as a donor, Aga2 was able to catalyze glycosyl transfer to various acceptors including monosaccharides, disaccharides and sugar alcohols.

Rapid burst kinetics in the hydrolysis of 4-nitrophenyl acetate by penicillin G acylase from Kluyvera citrophila. Effects of mutation F360V on rate constants for acylation and de-acylation

The kinetics of release of 4-nitrophenol were followed by stopped-flow spectrophotometry with two 4-nitrophenyl ester substrates of penicillin G acylase from Kluyvera citrophila. With the ester of acetic acid, but not of propionic acid, there was a pre-steady-state exponential phase, the kinetics of which were inhibited by phenylacetic acid (a product of hydrolysis of specific substrates) to the extent predicted from Ki values. This was interpreted as deriving from rapid formation (73 mM-1.s-1) and slow hydrolysis (0.76 s-1) of an acetyl derivative of the side chain of the catalytic-centre residue Ser-290. With the mutant F360V, which differs from the wild-type enzyme in its ability to hydrolyse adipyl-L-leucine and has a kcat for 4-nitrophenyl acetate one-twentieth that of the wild-type enzyme, the corresponding values for the rates of formation and hydrolysis of the acetyl-enzyme were 11.1 mM-1.s-1 and 0.051 s-1 respectively. The ratio of these rate constants was three times that for the wild-type enzyme, suggesting that the mutant is less impaired in the rate of formation of an acetyl-enzyme than in its subsequent hydrolysis.

RK7-kallikrein in rat kidney and urine

Rat Kidney is reported to contain the rK7-kallikrein gene expressed at a high mRNA level. However, there is no direct evidence that the rK7 enzyme is present in kidney or urine. We have analyzed rat kidney and urine for rK7-like enzyme activity, using distinctive properties of purified rK7 enzyme (hydrolysis of specific substrate, inhibition by SBTI). Rat urine and kidney cortex contain substantial levels of rK7-like enzyme activity. Nearly 40% of the enzyme in urine is in an active form while nearly all of the rK7 in kidney cortex is in the inactive form. Treatment with low salt diet and mineralocorticoid increases rK7 in both kidney cortex and urine approximately 2-fold. The percentage of active enzyme in urine increases markedly on this regimen.

Determination of acetylcholinesterase activity by gas-liquid chromatography

1. A modified Deans' method for quantitative gas-chromatographic determination of trace components in a large volume of sample of test mixture introduced into the apparatus was developed and used. 2. In the suggested modification this method can be used for gas-chromatographic determination of microquantities of acetic acid. 3. Deans' method can be used for quantitative differential determination of acetylcholinesterase and pseudocholinesterase in the blood, based on determination of end products of hydrolysis of specific substrates.

Metabolism in rats of 3‐phenoxybenzyl alcohol and 3‐phenoxybenzoic acid glucoside conjugates formed in plants

AbstractUpon single oral administration to rats, the mono‐, di‐ and tri‐glucose conjugates of [14C]‐3‐phenoxybenzyl alcohol (I) or the mono‐glucose conjugate of [14C]‐3‐phenoxybenzoic acid (II) were rapidly hydrolysed and extensively eliminated in the urine mostly as the sulphate conjugate of 3‐(4‐hydroxyphenoxy)benzoic acid (X). The faecal elimination was a minor route, whereas the biliary excretion was about 42% of the dose and the glucuronide conjugates of I, II and X were common major metabolites. The biliary glucuronides were cleaved in the small intestine to the respective aglycones, which were reabsorbed, metabolised further, and excreted in the urine as the sulphate conjugate of X. Although small amounts of the mono‐, di‐and tri‐glucosides were found in the 0.5‐h blood and liver samples following oral administration of the tri‐glucoside of I, they were not detected in the urine, bile or faeces. Similarly the sulphate conjugate was one of the major urinary metabolites of germ‐free rats, dosed with the 14C‐glucosides via the oral or the intraperitoneal route, although they were excreted unchanged in certain amounts in the urine and faeces. The glucose conjugates were cleaved in vitro by gut microflora and in various rat tissues, including blood, liver, small intestine and small intestinal mucosa. The tissue enzymes showed a different substrate specificity in hydrolysis of the glucosides. However, they were not cleaved in gastric juice, bile, pancreatic juice or urine.

Mechanism of action of cysteine proteinases: oxyanion binding site is not essential in the hydrolysis of specific substrates

To study the possible stabilization of the oxyanion of the tetrahedral intermediate formed in the course of the catalyses by cysteine proteinases, papain, chymopapain, papaya peptidase A, and ficin, we synthesized N-(benzyloxycarbonyl)phenylalanylthioglycine O-ethyl ester and compared its hydrolysis with that of the corresponding oxygen ester, a highly specific substrate of the above enzymes. It was found that the substitution of sulfur for the carbonyl oxygen hardly affected the second-order rate constant of acylation and diminished catalytic activity by about 1 order of magnitude in deacylation. These results contrast with those obtained with serine proteinases [Asbóth, B., & Polgár, L. (1983) Biochemistry 22, 117-122], where the hydrolysis of thiono esters could not be detected. From the results the following conclusions can be drawn. Stabilization of the tetrahedral intermediate at an oxyanion binding site is not essential with cysteine proteinases. Therefore, and because of the lack of general base catalysis, cysteine proteinases have a less constrained transition-state structure than serine proteinases.

Methanol-induced release of tightly bound adenine nucleotides from thylakoid-associated CF 1

Incubation of thylakoids in 33% methanol causes a release of the tightly bound nucleotides from CF 1. This methanol effect is not a stimulation of nucleotide exchange, since no medium ATP or ADP is incorporated into CF 1 during the methanol treatment. While the optimal conditions for stimulating the release of tightly bound ADP were similar to those for activating the ATPase, a direct relationship between the effects was not found. The tightly bound ADP does not represent a catalytic intermediate in this system, since (a) its rate of release is much slower than enzyme turnover, and (b) the substrate specificity for hydrolysis is different from that which promotes ADP release. A regulatory role for the tightly bound ADP in methanol-activated ATPase is also not indicated, since (a) activation of the ATPase occurs much more rapidly than ADP release, and (b) after the tightly bound ADP has been lost, high rates of ATP hydrolysis still require the presence of methanol, and (c) the small ATPase activity which persists after the removalo of the methanol is not correlated with the loss of bound ADP. These results show that significant rates of ATP hydrolysis can occur with ADP still tightly bound to CF 1. This argues against any model in which ADP regulates ATPase activity by binding directly to the catalytic site.

Solvent effects on the deacylation of acyl-chymotrypsins: a critical comment on the charge-relay hypothesis

The pH dependencies for deacylation of a series of acyl-chymotrypsins, prepared by using peptide-free enzyme, are reported. The pKa′ values exhibit considerable variation, from 7.63 for α-benzamido-trans-cinnamoyl-chymotrypsin to 6.6 for 3,5-dinitrobenzoyl-chymotrypsin. It is proposed that the variation results from an interaction of variable strength between the carboxylate ion of Asp-102 and protonated His-57 and, further, that the kinetically determined values of the pKa′ are attributable in all cases to the ionization of His-57. High values for the pKa′ controlling deacylation are therefore indicative of a strong electrostatic interaction and low values reflect a relatively weak interaction. The effect of 20.4% (w/w) dioxane on the pKa′' associated with the deacylation of a numher of acyl-chymotrypsins is reported. The measured shifts in the value of the pKa′ provide strong evidence for this description of the Asp-His pair in acyl-chymotrypsins. In the hydrolysis of the specific substrate N-acetyl-L-tryptophan ethyl ester, the ionizing group controlling deacylation behaves as a simple cationic acid. The results clearly indicate that a charge relay is not involved in the hydrolysis of specific substrates and argue against a kinetically important role for aspartic acid acting as a base in the mechanism of action of the serine proteinases.

Effect of methylamine on trypsin catalysis.

The effect of the basic group of specific substrates of trypsin on the kinetics of the enzyme was studied. The basic group was substituted by methylamine added to a non‐specific substrate. The kinetic constants of tryptic hydrolysis of N‐acetyl‐l‐alanine ethyl ester involving methylamine, at pH 6.3–8.5 were determined. For comparison, the constants of trypsin and α‐chymotrypsin were also determined.It can be concluded that methylamine increases the rate of hydrolysis catalyzed by trypsin. The relative increase of the pH‐independent limiting value of (kc/Km)max is shown to be fivefold and the pKa decreases by 0.4 unit. The increase of kc is nearly fourfold. The rate constants of the hydrolysis steps catalyzed by trypsin and trypsin · methylamine, respectively, were assayed by means of the nucleophile 1,4‐butanediol at pH 6.6. There is a sixfold increase of the original rates caused by methylamine for acylation (k2) and a threefold increase for deacylation (k3) but the substrate dissociation constant (Ks) was almost unchanged.A comparison of the individual rates of the methylamine‐activated tryptic hydrolysis of nonspecific substrates with the appropriate data of the trypsin‐catalyzed hydrolysis of specific substrates indicates that there is an approximately 1000‐fold difference between the rate constants for the acylation step, while in deacylation the difference is insignificant.The methylammonium ion bound to trypsin is supposed to keep the enzyme through the catalysis in a more active conformation than the original one. This indirect effect might bear only a small part of the trypsin‐substrate interaction.

Thiohemiacetal formation by inhibitory aldehydes at the active site of papain.

Papain is strongly inhibited by aldehydes resembling carboxylic acids, released by hydrolysis of specific substrates (Westerik, J. O'C., and Wolfenden, R. (1972), J. Biol. Chem. 247, 8195-8197). Inhibitory complexes might involve binding of the aldehyde intact or as a covalent hydrate, or the aldehyde might undergo covalent addition of an active site sulfhydryl group to form a thiohemiacetal derivative. In an attempt to distinguish between these possibilities, benzamidoacetaldehyde-1-d has been synthesized, and its properties compared with those of the undeuterated inhibitor. After correction for differences in hydration, the observed effect on inhibition is found to be compatible with formation of a thiohemiacetal. In keeping with this conclusion, benzamidoethanol (a partial analogue of the covalent hydrate) and benzamide, N-methylbenzamide and N-ethylbenzamide (somewhat similar to the free aldehyde in size and hydrophobic character) are found to exhibit negligible affinity for the active site.

Metal ions in enzymes using ammonia or amides.

In an attempt to understand the role of nickel in jack bean urease (1), we turned to a variety of other enzymes important in the utilization, production, or transfer of ammonia. We found several, including the L-histidine and L-phenylalanine ammonialyases and some enzymes that utilize glutamine or ammonia in amidotransferase reactions, all of which show evidence for the involvement of as yet unreported transition metal ions in their mechanism of action. We support the view that catalysis by metalloenzymes may be a reflection of the chemistry of the metal ion itself as a Lewis acid, and that perhaps too much emphasis has been placed on supposed special characteristics (such as strains, "entasis") of the enzyme-metal ion association. In this context, we have discussed the mechanism of catalysis of hydrolysis of specific substrates by carboxypeptidase A, and have returned to urease to examine the role of nickel in its mechanism of action.

Allosteric activation of the hydrolysis of specific substrates by chymotrypsin.

A variety of azobenzene compounds having bis-quaternary nitrogens have been shown to accelerate the hydrolysis by chymotrypsin of certain specific substrates by an allosteric mechanism. One of the most potent, 2,2'-bis[alpha-(benzyldimethylammonium)methyl]azobenzene dibromide (2,2'-QBzl) accelerated the hydrolysis of glutaryl-L-phenylalanine p-nitroanilide 40-fold at saturating concentration. Acceleration was by increasing kcat without altering Km. The hydrolysis of acetyl-L-tyrosine p-nitroanilide and acetyl-L-tyrosine anilide was also accelerated by Q-Bzl (25-fold and 1.8-fold respectively) while the hydrolysis of hemoglobin, azocoll and a number of esters was not affected. The inactivation of chymotrypsin by diphenylcarbamyl chloride and diphenylcarbamyl fluoride was accelerated by 2,2'-Q-Bzl. Reac;ivation in the presence of NH2OH was also accelerated, but in the absence of added nucleophile (i.e. of NH20H) no increase in rate was detectable. An allosteric effector was covalently attached to chymotrypsinogen A by reaction with 2,2'-bis[alpha-(o-bromomethylbenzyldimethylammonium)methyl]azobenezene dibromide. The product, when converted to active enzyme, was about 4 times more active than chymotrypsin as a result of an increase in kcat of hydrolysis; Km was unaffected. The mechanism of the allosteric acceleration process is not known but, because for all of the substrates affected acylation of the enzyme is rate-limitimg, it is tentatively suggested that the effectors facilitate proton transfer to the leaving group by an inductive effect on the 'charge relay system'. Spectral studies indicate that the allosteric site is a portion of the enzyme with a polarity near that of water, possibly on the outside surface of the enzyme molecule.

Allosteric activation of chymotrypsin-catalyzed hydrolysis of specific substrates

The rates of hydrolysis of three specific substrates of chymotrypsin, glutaryl-L-phenylalanine p-nitroanilide, acetyl-DL-tyrosine p-nitroanilide and acetyl-L-tyrosine anilide were enhanced by 2,2′-bis[α-(benzyldimethylammonium)methyl] azobenzene dibromide and less so by related compounds. Detailed studies with glutaryl-L-phenylalanine p-nitroanilide showed a 42-fold increase in k cat with no change in Km. No acceleration (or inhibition) was noted with esters, hydroxamides or proteins as substrates. Tryptic hydrolysis of benzoyl-DL-arginine p-nitroanilide was unaffected. It was concluded that certain quaternary compounds can act as allosteric effectors of chymotrypsin.