Kinetic, Catalytic and Thermodynamic Properties of Immobilized Milk Clotting Enzyme on Activated Chitosan Polymer and its Application in Cheese Making

Milk clotting enzyme (MCE) from Bacillus circulans 25 was immobilized by covalent binding, ionic binding and entrapment methods using various carriers. MCE covalently immobilized on activated chitosan polymer with the bifunctional agent glutaraldehyde (Ch-MCE) exhibited highest immobilization yield (74.6 %). Comparing to the native MCE, Ch-MCE exhibited higher optimum pH, higher optimum reaction temperature, lower activation energy, higher half-life time, lower deactivation rate constant and higher energy for denaturation. After immobilization, maximum reaction rate, Michaelis-Menten constant, specificity constant, turnover number, and catalytic efficiency of the enzyme were significantly changed. Calculated thermodynamic parameters for denaturation (enthalpy, entropy and Gibbs free energy) confirmed that the catalytic properties of MCE were significantly improved after immobilization. Reusability tests showed that after 7 catalytic cycles, the Ch-MCE retained about 71 % of its activity confirming its suitability for industrial applications.

Antithrombin resistance rescues clotting defect of homozygous prothrombin-Tyr510N dysprothrombinemia

A patient with hematuria in our clinic was diagnosed with urolithiasis. Analysis of the patient’s plasma clotting-time indicated that both APTT (52.6 s) and PT (19.4 s) are prolonged and prothrombin activity is reduced to 12.4% of normal, though the patient exhibited no abnormal bleeding phenotype and a prothrombin antigen level of 87.9%. Genetic analysis revealed the patient is homozygous for prothrombin Y510N mutation. We expressed and characterized the prothrombin-Y510N variant in appropriate coagulation assays and found that the specificity constant for activation of the mutant zymogen by factor Xa is impaired ~5-fold. Thrombin generation assay using patient’s plasma and prothrombin-deficient plasma supplemented with either wild-type or prothrombin-Y510N revealed that both peak height and time to peak for the prothrombin mutant are decreased however the endogenous thrombin generation potential is increased. Further analysis indicated that the thrombin mutant exhibits resistance to antithrombin and is inhibited by the serpin with ~12-fold slower rate constant. Protein C activation by thrombin-Y510N was also decreased ~10-fold, however, thrombomodulin overcame the catalytic defect. The Na+-concentration-dependence of the amidolytic activities revealed that the dissociation constant for the interaction of Na+ with the mutant has been elevated ~20-fold. These results suggest that Y510 (Y184a in chymotrypsin numbering) belongs to network of residues involved in binding Na+. A normal protein C activation by thrombin-Y510N suggests that thrombomodulin modulates the conformation of the Na+-binding loop of thrombin. The clotting defect of thrombin-Y510N appears to be compensated by its markedly lower reactivity with antithrombin, explaining patient’s normal hemostatic phenotype.

Reversible enzyme-catalysed reactions: the quasi steady state as a a quasi-equilibrium, and a correction to Haldane's kinetic constants and to secondary relationships involving kinetic constants.

For reversible enzyme-catalysed reactions obeying Henri-Michaelis-Menten kinetics, theoretical solution of the rate equations for the enzyme-substrate intermediate are generally incorrect when the quasi-steady state approximation, equating the rate of change of the concentration of the enzyme-substrate intermediate to zero, is used. For the simplest kinetic model used by Haldane, such a procedure does not reveal that in one direction, that starting with the reactant having the lower binding constant, the quasi-steady state is one of quasi-equilibrium, and Haldane’s structure of the Km written in terms of rate constants is incorrect. This is probably also true of more complex mechanisms in which the structure of kcat may also be in error. Modern methods of numerical integration for the solution of rate equations, if applied to reversible reactions to obtain rate constants from measured catalytic constants, will require the correct expressions for kcat and Km. Furthermore, the (now called) Haldane relationship, relating the kinetic constants kcat and Km for the forward and reverse reactions to the equilibrium constant of a reaction, is now seen to be generally incorrect, and in addition another exception for a the theoretical validation of kcat /Km as a specificity constant arises.

Universal capability of 3-ketosteroid Δ1-dehydrogenases to catalyze Δ1-dehydrogenation of C17-substituted steroids

Background 3-Ketosteroid Δ1-dehydrogenases (KSTDs) are the enzymes involved in microbial cholesterol degradation and modification of steroids. They catalyze dehydrogenation between C1 and C2 atoms in ring A of the polycyclic structure of 3-ketosteroids. KSTDs substrate spectrum is broad, even though most of them prefer steroids with small substituents at the C17 atom. The investigation of the KSTD’s substrate specificity is hindered by the poor solubility of the hydrophobic steroids in aqueous solutions. In this paper, we used 2-hydroxpropyl-β-cyclodextrin (HBC) as a solubilizing agent in a study of the KSTDs steady-state kinetics and demonstrated that substrate bioavailability has a pivotal impact on enzyme specificity. Results Molecular dynamics simulations on KSTD1 from Rhodococcus erythropolis indicated no difference in ΔGbind between the native substrate, androst-4-en-3,17-dione (AD; − 8.02 kcal/mol), and more complex steroids such as cholest-4-en-3-one (− 8.40 kcal/mol) or diosgenone (− 6.17 kcal/mol). No structural obstacle for binding of the extended substrates was also observed. Following this observation, our kinetic studies conducted in the presence of HBC confirmed KSTD1 activity towards both types of steroids. We have compared the substrate specificity of KSTD1 to the other enzyme known for its activity with cholest-4-en-3-one, KSTD from Sterolibacterium denitrificans (AcmB). The addition of solubilizing agent caused AcmB to exhibit a higher affinity to cholest-4-en-3-one (Ping-Pong bi bi KmA = 23.7 μM) than to AD (KmA = 529.2 μM), a supposedly native substrate of the enzyme. Moreover, we have isolated AcmB isoenzyme (AcmB2) and showed that conversion of AD and cholest-4-en-3-one proceeds at a similar rate. We demonstrated also that the apparent specificity constant of AcmB for cholest-4-en-3-one (kcat/KmA = 9.25∙106 M−1 s−1) is almost 20 times higher than measured for KSTD1 (kcat/KmA = 4.71∙105 M−1 s−1). Conclusions We confirmed the existence of AcmB preference for a substrate with an undegraded isooctyl chain. However, we showed that KSTD1 which was reported to be inactive with such substrates can catalyze the reaction if the solubility problem is addressed.

Universal Capability of 3-Ketosteroid Δ1-Dehydrogenases to Catalyze Δ1-Dehydrogenation of C17- Substituted Steroids

Background 3-Ketosteroid Δ1-dehydrogenases (KSTDs) are the enzymes involved in microbial cholesterol degradation and modification of steroids. They catalyze dehydrogenation between C1 and C2 atoms in ring A of the polycyclic structure of 3-ketosteroids. KSTDs substrate spectrum is broad, even though most of them prefer steroids with small substituents at the C17 atom. The investigation of the KSTD’s substrate specificity is hindered by the poor solubility of the hydrophobic steroids in aqueous solutions. In this paper, we used 2-hydroxpropyl-β-cyclodextrin (HBC) as a solubilizing agent in a study of the KSTDs steady-state kinetics and demonstrated that substrate bioavailability has a pivotal impact on enzyme specificity. Results Molecular dynamics simulations on KSTD1 from Rhodococcus erythropolis indicated no difference in ΔGbind between the native substrate, androst-4-en-3,17-dione (AD; − 8.02 kcal/mol), and more complex steroids such as cholest-4-en-3-one (–8.40 kcal/mol) or diosgenone (–6.17 kcal/mol). No structural obstacle for binding of the extended substrates was also observed. Following this observation, our kinetic studies conducted in the presence of HBC confirmed KSTD1 activity towards both types of steroids. We have compared the substrate specificity of KSTD1 to the other enzyme known for its activity with cholest-4-en-3-one, KSTD from Sterolibacterium denitrificans (AcmB). The addition of solubilizing agent caused AcmB to exhibit a higher affinity to cholest-4-en-3-one (Ping-Pong bi bi KmA= 23.7 µM) than to AD (KmA= 529.2 µM), a supposedly native substrate of the enzyme. Moreover, we have isolated AcmB isoenzyme (AcmB2) and showed that conversion of AD and cholest-4-en-3-one proceeds at a similar rate. We demonstrated also that the apparent specificity constant of AcmB for cholest-4-en-3-one (kcat/KmA= 9.25 ∙ 106 M− 1 s− 1) is almost 20 times higher than measured for KSTD1 (kcat/KmA= 4.71 ∙ 105 M− 1 s− 1). Conclusions We confirmed the existence of AcmB preference for a substrate with an undegraded isooctyl chain. However, we showed that KSTD1 which was reported to be inactive with such substrates can catalyze the reaction if the solubility problem is addressed.

Comparative biochemistry of four polyester (PET) hydrolases

The potential of bioprocessing in a circular plastic economy has strongly stimulated research in enzymatic degradation of different synthetic resins. Particular interest has been devoted to the commonly used polyester, poly(ethylene terephthalate) (PET), and a number of PET hydrolases have been described. However, a kinetic framework for comparisons of PET hydrolases (or other plastic degrading enzymes) acting on the insoluble substrate, has not been established. Here, we propose such a framework and test it against kinetic measurements on four PET hydrolases. The analysis provided values of kcat and KM, as well as an apparent specificity constant in the conventional units of M−1s−1. These parameters, together with experimental values for the number of enzyme attack sites on the PET surface, enabled comparative analyses. We found that the PET hydrolase from Ideonella sakaiensis was the most efficient enzyme at ambient conditions, and that this relied on a high kcat rather than a low KM. Moreover, both soluble and insoluble PET fragments were consistently hydrolyzed much faster than intact PET. This suggests that interactions between polymer strands slow down PET degradation, while the chemical steps of catalysis and the low accessibility associated with solid substrate were less important for the overall rate. Finally, the investigated enzymes showed a remarkable substrate affinity, and reached half the saturation rate on PET, when the concentration of attack sites in the suspension was only about 50 nM. We propose that this is linked to nonspecific adsorption, which promotes the nearness of enzyme and attack sites.

Characterization of the Novel Ene Reductase Ppo-Er1 from Paenibacillus Polymyxa

Ene reductases enable the asymmetric hydrogenation of activated alkenes allowing the manufacture of valuable chiral products. The enzymes complement existing metal- and organocatalytic approaches for the stereoselective reduction of activated C=C double bonds, and efforts to expand the biocatalytic toolbox with additional ene reductases are of high academic and industrial interest. Here, we present the characterization of a novel ene reductase from Paenibacillus polymyxa, named Ppo-Er1, belonging to the recently identified subgroup III of the old yellow enzyme family. The determination of substrate scope, solvent stability, temperature, and pH range of Ppo-Er1 is one of the first examples of a detailed biophysical characterization of a subgroup III enzyme. Notably, Ppo-Er1 possesses a wide temperature optimum (Topt: 20–45 °C) and retains high conversion rates of at least 70% even at 10 °C reaction temperature making it an interesting biocatalyst for the conversion of temperature-labile substrates. When assaying a set of different organic solvents to determine Ppo-Er1′s solvent tolerance, the ene reductase exhibited good performance in up to 40% cyclohexane as well as 20 vol% DMSO and ethanol. In summary, Ppo-Er1 exhibited activity for thirteen out of the nineteen investigated compounds, for ten of which Michaelis–Menten kinetics could be determined. The enzyme exhibited the highest specificity constant for maleimide with a kcat/KM value of 287 mM−1 s−1. In addition, Ppo-Er1 proved to be highly enantioselective for selected substrates with measured enantiomeric excess values of 92% or higher for 2-methyl-2-cyclohexenone, citral, and carvone.

New standards for collecting and fitting steady state kinetic data

The Michaelis–Menten equation is usually expressed in terms of k cat and K m values: v = k cat[S]/(K m + [S]). However, it is impossible to interpret K m in the absence of additional information, while the ratio of k cat/K m provides a measure of enzyme specificity and is proportional to enzyme efficiency and proficiency. Moreover, k cat/K m provides a lower limit on the second order rate constant for substrate binding. For these reasons it is better to redefine the Michaelis–Menten equation in terms of k cat and k cat/K m values: v = k SP[S]/(1 + k SP[S]/k cat), where the specificity constant, k SP = k cat/K m. In this short review, the rationale for this assertion is explained and it is shown that more accurate measurements of k cat/K m can be derived directly using the modified form of the Michaelis–Menten equation rather than calculated from the ratio of k cat and K m values measured separately. Even greater accuracy is achieved with fitting the raw data directly by numerical integration of the rate equations rather than using analytically derived equations. The importance of fitting to derive k cat and k cat/K m is illustrated by considering the role of conformational changes in enzyme specificity where k cat and k cat/K m can reflect different steps in the pathway. This highlights the pitfalls in attempting to interpret K m, which is best understood as the ratio of k cat divided by k cat/K m.

DNA unwinding is the primary determinant of CRISPR-Cas9 activity

SummaryBacterial adaptive immunity utilizes RNA-guided surveillance complexes composed of CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target foreign nucleic acids for destruction. Cas9, a type II CRISPR-Cas effector complex, can be programed with a single guide RNA that base-pairs with the target strand of dsDNA, displacing the non-target strand to create an R-loop, where the HNH and RuvC nuclease domains can cleave opposing strands. Cas9 has been repurposed for a variety of important genome engineering applications. While many structural and biochemical studies have shed light on the mechanism of Cas9 cleavage, a clear unifying model has yet to emerge. Our detailed kinetic characterization of the enzyme reveals that DNA binding is reversible, R-loop formation is rate-limiting, occurring in two steps, one for each of the nuclease domains. Although the HNH nuclease activity is stimulated by Mg2+ with a single measureable Kd, the RuvC activity requires two distinct Mg2+ binding events. The specificity constant for cleavage is determined through an induced-fit mechanism as the product of the equilibrium binding affinity for DNA and the rate of R-loop formation.