Substituent effects on substrate activation and Michaelis-Menten Kinetic parameters in the alpha-chymotrypsin-catalyzed hydrolysis of phenyl acetates.

The effects of substituents on the steady state and pre-steady state kinetics in alpha-chymotrypsin [EC 3.4.21.1]-catalyzed hydrolysis were studied using substituted phenyl acetates. In the steady state hydrolysis, substrate activation, which had been observed and studied previously for p-nitrophenyl acetate, was also observed for p-bromo, p-chloro-, and m-methylphenyl acetates. Little activation was observed for p-acetyl-, m-nitro-, p-methyl-, and p-methoxyphenyl acetates. Addition of p-dichlorobenzene increased kcat for all substrates examined and greatly diminished the substrate activation for the activatable substrate(s) to activator binding site(s). The value of kcat decreased in accordance with increase of the sigma-value of substituents. On the other hand, kcat/Km (app) showed an opposite sigma- dependence, as was previously observed. In pre-steady state measurements, little burst was observed for more electron-donating substituents than m-nitro. The sigma dependence of kcat is apparently not consistent with the prediction derived from that of kcat/Km (app) on the basis of the usual two-step mechanism with a common acetyl-enzyme intermediate.     

A rate determining step change in the pre-steady state of acetylcholinesterase inhibitions by 1,n-alkane-di-N-butylcarbamates

Alkane-1-N-butylcarbamate-n-ols (1-7) and 1,n-alkane-di-N-butylcarbamates (8-14) are potent pseudo-substrate inhibitors of acetylcholinesterase. For inhibitors 1-7, the pre-steady state -logK(s) values and steady state -logK(i), values are linearly correlated with the tether length (N). However, for inhibitors 8-14, correlation of the -logK(s) or -logK(i) values against N deviates from linearity. A discontinuity of the -logK(s) versus N plot, concave downwards, is indicative of a rate determining step change in the pre-steady state of acetylcholinesterase inhibitions by inhibitors 8-14.     

Enthalpy of acetylcholine hydrolysis by acetylcholinesterase

Direct microcalorimetric measurements were made of the reaction between acetylcholine chloride and acetylcholinesterase (EC 3.1.1.7) that was extracted from electric eel (Electrophorus electricus) and purified by affinity chromatography. Tris-HCl, sodium phosphate and potassium phosphate were used as buffers and sources of ions for the reaction. At pH 7.2 and in 0.1-0.2 M phosphate buffer, the delta H for acetylcholine hydrolysis was found to be -0.107 kcal/mol (under buffered conditions) and -0.931 kcal/mol under unbuffered conditions (water). At pH 8.0 in 0.1 M Tris-HCl buffer, values greater than -2.5 kcal/mol were obtained, with the highest value of -9.2 kcal/mol being seen with bovine erythrocyte acetylcholinesterase. Tris-HCl buffer at 4 X 10(-2) M enhanced the reaction velocity by 51.2% over that of 4 X 10(-3) M buffer. Enzyme purity, pH and ionic milieu of reaction mixture, and substrate concentration affected the measured delta H value.     

Enzyme mediated hydrolysis of acetates

Summary The investigation of enzyme mediated hydrolysis of the respective cis- and trans-2-(4-methoxybenzyl)-1-cyclohexyl acetates has been provided using several lipases. A comparison of results obtained has been summarized in this paper.     

Serine activation is the rate limiting step of tRNASer aminoacylation by yeast seryl tRNA synthetase.

Using the quenched flow technique the mechanism of seryl tRNA synthetase action has been investigated with respect to the presteady state kinetics of individual steps. Under conditions where the strong binding sites of the enzyme are nearly saturated and the steady state turnover number is about 1 s-1, rate constants of four different processes have been determined: steps connected with substrate associations are relatively slow (12 s-1 for the entire process); activation of serine is the rate determining step (about 1.2 s-1 in presence of tRNASer); whereas the transfer of serine onto tRNASer (35 s-1) and the dissociation of seryl tRNASer (70 s-1) are fast. Similar kinetic parameter seem to hold also for the steady state reactions. This conclusion is based on a detailed study of the substrate, product, and Mg2+ concentration dependence of the transfer reaction. The results also indicate that a second serine binding site is operative. Since the transfer of serine from a preformed adenylate complex onto tRNASer is fast, seryl adenylate seems to be a kinetically competent intermediate of the aminoacylation reaction although, of course, alternative mechanisms cannot be excluded.     

On the rate limiting step in downhill transport via the LacY permease of Escherichia coli

Strains of Escherichia coli K12 were constructed for the specific purpose of evaluating the inducibility of the influx mechanism controlled by the lacY gene. These strains are heteromerodiploids characterized by a high and relatively constant level of β-D-galactosidase which is not affected significantly by induction of the Lac operon. These properties were obtained by introducing episomal lacI⁺,O^c,Z⁺,Y^? genes into the cells. In these merodiploids the rate of o-nitrophenyl-β-D-galactopyranoside (ONPG) hydrolysis of extracted cells is 50-times that of intact cells. This difference indicates that the rate limiting step in the ONPG hydrolysis by intact cells is influx. Using a set of merodiploids with and without the LacY transport system, we were able to demonstrate a specific induction of ONPG influx. However, the increase in influx due to induction was only 3.5-fold as compared to the 40-fold increase observed when the LacY permease was measured by intracellular accumulation of [¹⁴C] TMG.     

Factors limiting the hydrolysis of casein by subtilisin DY

It was shown that during the subtilisin DY-induced hydrolysis of casein relatively stable polypeptide structures are formed. In their interior these structures contain peptide bonds which are susceptible to the enzyme used. Heating (up to 100 degrees C) and/or application of ultrasound (25 kHz, 60 W) results in their unfolding. Data are provided, which show that under the enzyme-substrate complex formation does not lead to an enzyme conformation more susceptible to autolysis. Taking into account the described phenomena a higher degree of hydrolysis was attained in comparison to those obtained by standard enzymatic hydrolysis.     

Computational modeling of the rate limiting step in low molecular weight protein tyrosine phosphatase.

Hydrolysis of the phosphoenzyme intermediate is the second and rate limiting step of the reaction catalyzed by the protein tyrosine phosphatases (PTPs). The cysteinyl phosphate thioester bond is cleaved by nucleophilic displacement where an active site water molecule attacks the phosphorus atom. Starting from the crystal structure of the low molecular weight PTP, we study the energetics of this reaction utilizing the empirical valence bond method in combination with molecular dynamics and free energy perturbation simulations. The reactions of the wild-type as well as the D129A and C17S mutants are modeled. For the D129A mutant, which lacks the general acid/base residue Asp-129, an alternative reaction mechanism is proposed. The calculated activation barriers are in all cases in good agreement with experimental reaction rates. The present results together with earlier computational and experimental work now provide a detailed picture of the complete reaction mechanism in many PTPs. The key role played by the structurally invariant signature motif in stabilizing a double negative charge is reflected by its control of the energetics of both transition states and the reaction intermediate.     

Pre-treatments to enhance the biodegradability of waste activated sludge: Elucidating the rate limiting step

Pre-treatments for waste activated sludge (WAS) are, in most cases, an attempt to increase the biodegradation and/or improve hydrolysis rate of WAS after anaerobic digestion. This review presents an extensive analysis of WAS pre-treatments effectiveness focusing on increasing the biodegradability. In the first part of the review, WAS is considered as a cluster of organic components: proteins, carbohydrates, humic substances and cells. Based on this breakdown into components, the effect of different pre-treatments on each component (and in combination) is described. Also, possible reasons for the contradictory results frequently found among different studies dealing with the same pre-treatment are included. In the second part, the review describes the effects on volatile solids removal by digestion after pre-treatment and on the dewaterability of the final digestate. The energy balance and potential limiting factors for each pre-treatment are also taken into account. From the published works it is concluded that some pre-treatment techniques, such as thermal hydrolysis, thermal phased anaerobic digestion and low-temperature pre-treatment are effective ways to increase energy production and to improve other sludge properties, such as dewatering. However, these techniques are very energy intensive and require a large capital outlay, so research on milder pre-treatment techniques is valuable.     

Bacillus subtilisα-amylase: the rate limiting step of secretion is growth phase-independent

When Bacillus subtilis alpha-amylase was expressed under the control of sacR in a degU32(Hy) strain, the production of exoenzyme occurred during both the exponential and stationary phases of growth. In each phase, pulse-chase experiments showed that the rate-limiting step of the secretion process was the release of the processed form of the protein in each physiological context. The rate of this event was slightly slower (t(1/2) = 3.2 min) during the stationary phase than during the exponential phase (t(1/2) = 2 min). The effectors which possibly control the efficiency of the release stage, the level of PrsA or the calcium binding properties of the cell wall, remained unchanged throughout growth phases.     

Regioselective hydrolysis of acetates in the presence of different yeast strains

The model compound, hexane-1,2-diol diacetate, was hydrolyzed in the presence of supernatant obtained after cultivation of 4 yeast strains: Pichia jadinii, Rhodotorula glutinis and Yarrowia lipolytica KKP 379 and Saccharomyces cerevisiae 102 to evaluate the type of catalysis. The regioselectivity of extracellular enzymes as a function of hydrolysis towards primary and secondary acetic acid ester groups was monitored. The enzymes secreted by P. jadinii, R. glutinis and Y. lipolytica KKP 379 exhibited high regioselectivity towards primary position, while those from S. cerevisiae showed practically no discrimination between the ester groups.     

Inhibition of acetylcholinesterase hydrolysis of cationic substrates by the reaction product

The kinetics of acetylcholinesterase-catalyzed hydrolysis of the two cationic substrates (I and II in Russian text) was analyzed by means of the integrated Michaelis equation (3). The constants kII, kcat Km and the enzyme-product complex dissociation constant Ki were determined. (Table 1). It was shown that acetylcholine (II) binds to to the enzyme active center more effectively than the alcohol product of its hydrolysis. In case of the pipecholine derivative (I) reversed situation occurs. The different dependence of the ester substrate and appropriate alcohol binding effectiveness upon the reagent structure indicates the dissimilar location of the molecules in the active center of acetylcholinesterase. Some structural implications of the enzyme active center were discussed.     

A method for analyzing enzyme kinetics with substrate activation and inhibition and its application to the alpha-chymotrypsin-catalyzed hydrolysis of phenyl acetates.

A general kinetic method was developed to analyze enzyme-catalyzed systems complicated by the presence of activation or inhibition by substrate. The method was applied to the alpha-chymotrypsin [EC 3.4.21.1]-catalyzed hydrolysis of p-chlorophenyl and p-methoxyphenyl acetates. Deacylation rate constants which were not complicated by substrate activation were obtained. The analysis shows that the abnormal substituent dependence of kcat in the steady state hydrolysis is due not to substrate activation but to inappropriateness of the two-step mechanism or the existence of more than one acetyl-enzyme intermediate.