A theoretical study on the hydrolysis mechanism of carbon disulfide

The hydrolysis mechanism of CS(2) was studied using density functional theory. By analyzing the structures of the reactant, transition states, intermediates, and products, it can be concluded that the hydrolysis of CS(2) occurs via two mechanisms, one of which is a two-step mechanism (CS(2) first reacts with an H(2)O, leading to the formation of the intermediate COS, then COS reacts with another H(2)O, resulting in the formation of H(2)S and CO(2)). The other is a one-step mechanism, where CS(2) reacts with two H(2)O molecules continuously, leading to the formation of H(2)S and CO(2). By analyzing the thermodynamics and the change in the kinetic function, it can be concluded that the rate-determining step involves H and OH in H(2)O attacking S and C in CS(2), respectively, causing the C=S double bond to change into a single bond. The two mechanisms are competitive. When performing the hydrolysis of CS(2) with a catalyst, the optimal temperature is below 252°C.     

Reaction mechanism, specificity and pH-dependence of peptide synthesis catalyzed by the metalloproteinase thermolysin

The initial rates of peptide bond formation catalyzed by the metalloproteinase thermolysin were determined. The dependence of the formation rates on the concentration of the carboxyl donor and the acceptor can be explained by a rapid-equilibrium random bireactant mechanism, in which the binding of one substrate has a positive influence on the binding of the other (synergism). The specificity of the enzyme for the donor and acceptor in the condensation reaction was further investigated by determining the apparent kinetic parameters kcat and Km for various substrates. The pH-dependence of the initial rates of synthesis was found to be identical to the pH-dependence of the hydrolytic action of the enzyme. The rates are also shown to be independent of the pKa of the amino group of the acceptor, indicating that deprotonation of the attacking nucleophile in the synthetic reaction is not rate-limiting.     

A theoretical study of the attenuation control mechanism

The attenuator control mechanism, used in a number of amino acid biosynthetic operons, is considered from a theoretical point of view. The physics of RNA hairpin-loop formation is discussed, and rules for predicting which codons in the leader peptide that will affect operon expression are suggested. Manabe's (1981) stochastic model for the attenuator mechanism is used to analyse a number of known attenuators, showing a need for a “polymerase pause-site” in most of the attenuators, and providing some quantitative arguments in favour of the use of unusual codons in the control region.     

The hydrolysis by thermolysin of dipeptide derivatives that contain substituted cysteine

1. The preparation of protected dipeptides of the form acetylglycylamino acid amides is described, where the amino acid is phenylalanine, leucine, valine, alanine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine and S-phenylcysteine. 2. Kinetic parameters for the thermolytic hydrolysis of these blocked dipeptides are reported. The rate of hydrolysis was fastest when the amino acid was leucine or phenylalanine, slower when it was S-methylcysteine, valine or S-ethylcysteine, much slower when it was alanine, and negligible for S-phenylcysteine or S-benzylcysteine. 3. The results are compared with those for similar dipeptide derivatives with benzyloxycarbonyl and furylacryloyl blocking groups, which are hydrolysed faster.     

Enzymic hydrolysis of lignocellulosic materials: I. Models for the hydrolysis process--a theoretical study

At the end of an enzymic hydrolysis process involving a solid lignocellulosic substrate, enzymes are found both in solution and absorbed to the substrate residue. Removal of residue from the system will result in loss of some of the enzymes, the extent of which will depend on the design of the process. To minimize enzyme loss, a study has been conducted in which six process models have been formulated and an enzyme loss function derived for each model based on the total amount of enzymes lost through residue removal. Model 1 is a reference model, characterized by an uninterrupted hydrolysis throughout the entire hydrolysis period. The residue is then washed in order to recover both sugar and adsorbed enzymes before the residue is discarded. Models 2-6 are all characterized by the removal of hydrolysate three times during the process, recirculation of dissolved and adsorbed enzymes to various points in the process and selection of a stage at which the residue is removed. The following conclusions could be drawn from the derived enzyme loss functions: Increased enzyme adsorption leads to increased enzyme loss.The enzyme loss decreases if the solid residue is removed late in the process.Both adsorbed and dissolved enzymes should be introduced at the starting point of the process. This is particularly important for dissolved enzymes. Three models were chosen for experimental studies, which are reported in a second, accompanying article. The experimental results obtained are compared with the theoretical study reported here.     

The unfolding mechanism of thermolysin

The ligand-modulated kinetics of the autoproteolysis of thermolysin and the high-molecular-weight products of the reaction provide evidence for the conclusion that separation of the two structural domains is most probably the first step on the unfolding pathway of the protein under native conditions.     

Lanthanide-based fluorogenic peptide substrate for the highly sensitive detection of thermolysin

A new fluorogenic, lanthanide-based oligopeptide substrate for the detection of the zinc-dependent endoprotease thermolysin is described. Using time-resolved fluorescence measurement, a highly sensitive assay for thermolysin was developed with a 50 pM detection limit (3.5 fmol).     

The pH dependence of peptide hydrolysis by nitrocarboxypeptidase A

Hydrolysis of benzyloxycarbonyl-GlyGlyPhe by nitro(Tyr 248)carboxypeptidase A over the pH range 4.88–8.04 has been examined. The nitroenzyme retains appreciable activity near pH 6.5, and the limiting value of K_m is scarcely affected. The peptidase activity has a pH dependence characterized by the following parameters: pK_E1 of 6.37 ± 0.19 and pK_E2 of 6.60 ± 0.17 in $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$, and apparent pK of 5.59 ± 0.06 in K_cat. A spectroscopic pK of 6.75 ± 0.01, attributable to the nitro-Tyr 248 residue, has been determined. This correlates with the base-limb pK_E2 in the $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ profile, which appears to be shifted from a higher value, pK_E2 of 9.0, for the native enzyme. The single (acid-limb) pK which characterizes the k_cat profile of the native enzyme is also found to be perturbed to a lesser extent by nitration. A kinetically competent reverse protonation mechanism, based on chemical modification and crystallographic evidence for the enzyme, is described.     

The hydrolysis of 3-(2-furylacryloyl)-glycyl-l-leucine amide by thermolysin

The kinetics of the hydrolysis of 3-(2-furylacryloyl)-glycycl-l-leucine amide by thermolysin has been reinvestigated. It was found that the K_m for the enzyme substrate interaction is 2.5 × 10^?3m at pH 7.2. This K_m is an order of magnitude less than what has been previously assumed to be the K_m for the enzyme-substrate interaction. The normally recommended assay has 1–3 × 10^?3m substrate and is based on the assumption that the substrate concentration is much less than the K_m. Our data indicate that this assumption appears to be invalid. The hydrolysis of 3-(2-furylacryloyl)-glycyl-l-leucine amide results in a maximum decrease in absorbance at 322 nm. The change in absorbance is nearly 10-fold greater at 322 nm than the change in absorbance at 345 nm where the hydrolysis has been customarily followed. By following the hydrolysis of the substrate at 10^?4m at 322 nm it is possible to work under conditions where the substrate concentration is much less than the K_m.     

A mechanism for the hydrolysis of MgATP by actomyosin of skeletal muscle.

Based on a model of the active site of myosin (Ramirez, Shukla &; Levy, 1978), a chemical mechanism for MgATPase and intermediate oxygen exchange is presented. In this mechanism, oxygen exchange takes place via an oxyphosphorane intermediate that undergoes double turnstile rotation (Ugi, Ramirez, Marquarding, Klusacek &; Gillespie, 1971; Ramirez &; Ugi, 1974. During hydrolysis by native skeletal muscle myosin, only three [¹⁸O] atoms from labelled water are rapidly incorporated into the phosphorus that is finally released to the medium as P_i; whereas, during hydrolysis by subfragment 1 (S1), which is the head of myosin, four oxygens are labelled rapidly. To explain this difference, we postulate that cleavage of the (S1)-(S2) hinge in the preparation of S1 modifies the interaction of the oxyphosphorane intermediate at the active site. This enables a normally non-exchangeable oxygen to enter the exchange process. This is consistent with our earlier interpretation to the effect that the active site and the hinge in myosin are relatively close to each other Shukla &; Levy, 1977b; Shukla &; Levy, 1978. We postulate that the major elements of the active site are situated on a 92 amino acid fragment, p₁₀, isolated by Elzinga &; Collins, 1977 from myosin. P₁₀ is now known to be situated in the region that connects the head to the body of a myosin heavy chain (Lu, Sosinki, Balint &; Streter, 1978). An examination of the p₁₀ fragment for a possible point of proteolytic attack in the region of the hinge which will generate S1 revealed lysine 82. Breaking the protein chain at a point so close to the active site pocket could explain the effect of hinge cleavage on oxygen exchange. Two additional features of the present mechanism are: (1) the protonation of P_γ of a MgP_α,P_γ complex of ATP, which depresses monomeric metaphosphate mediated hydrolysis, and enhances oxyphosphorane formation by addition of water to P_γ; (2) the coordination of N^τ-methylhistidinet² of actin with Mg at the active site, which activates the release of the products of hydrolysis.     

The study of peptide stability during hydrolysis by rat gastroenteric tract fragments

The hydrolytic stability of therapeutic peptides such as dalargin, stemokin and some others, including cyclic tripeptides modified by ibuprofen and aspirin, was studied. Two experimental systems were used, one containing purified enzymes pepsin, trypsin and chymotrypsin and other based on fragments of rat stomach and ileum. It was found that linear peptides without D-aminoacids are hydrolyzed by fragments of stomach and ileum but resistant to hydrolysis with purified enzymes. The peptides with D-aminoacids and cyclic peptides are stable in all experimental conditions used, however, peptides modified with aspirin lost acetyl moiety of aspirin residue in acidic medium, the process is accelerated in presence of pepsin.     

A suicide-substrate mechanism for hydrolysis of beta-lactams by an anti-idiotypic catalytic antibody

The catalytic mechanism of an anti-idiotypic antibody, 9G4H9, displaying a beta-lactamase activity was investigated. Kinetics experiments suggest that some penicillinic derivatives behave both as substrates and inactivators. Biochemical and immunological experiments strongly indicate that ampicillin may be regarded as a suicide substrate for hydrolysis by 9G4H9. The anti-idiotypic network appears as a way to create enzyme mimics with modified catalytic activities.     

Fructose production by chicory inulin enzymatic hydrolysis: A kinetic study and reaction mechanism

In this paper, a reaction scheme for fructose production by inulin enzymatic hydrolysis is proposed, taking into account the possibility of dealing with a mixture of enzymes acting on a mixture of polymers as substrate. The scheme is subsequently simplified to obtain a stoichiometric relationship between the fructose product and the reacted substrate. The former may be measured by HPLC, while the latter is the subject of kinetic investigations. Our proposed kinetic model is defined within temperature and substrate concentration ranges of industrial interest (40–60 °C and 3–60 g/L, respectively). Some assumptions were made in order to simplify the model, which is based on a minimum number of parameters. These hypotheses were always specified and assumed only on the basis of convenience and rational consideration. Eventually, the kinetic model was successfully validated by comparison with a vast set of experimental results.