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Enzyme activators lower Km (the Michaelis constant) and/or raise Vmax (the asymptotic reaction velocity at infinite substrate concentration); conversely, inhibitors raise Km and/or lower Vmax . But what if an effector moves both Km and Vmax in the same direction? Uncompetitive inhibitors, which decrease both Km and Vmax by the same factor, are the most common example of this. A less well-known example occurs often when crowding agents are added to the buffer in order to mimic the environment commonly encountered in vivo. Crowding agents tested on different enzymes have been shown to have every conceivable effect on Km or Vmax , causing them to rise, fall, or stay the same. In this article, a mathematical analysis is presented allowing biochemists to judge whether an effector that causes Km and Vmax to both move in the same direction serves as an inhibitor, an activator, or most surprising, as both. © 2019 International Union of Biochemistry and Molecular Biology, 47(4):446-449, 2019.

As part of a study to investigate metal chelates as possible inhibitors of acetylcholinesterase, copper chelates of 1 ,3 diaminopropanol-2, 1,3 diaminopropane , and hydroxy-1-proline are examined. Since the chelate s olutions contain mixtures of species in equilibrium, it is necessary to calculate the concentrations of pertinent species under experimental conditions . Equations are derived which can be used to calculate the concentrations of individual species in an equilibrium mixture of ligand and copper under fixed conditions of concentration and pH with the aid of an IBM 1410 computer. Initial measurements of reaction velocity of enzyme-substrate breakdown to enzyme plus acid products, are made by observing the speed of pH drop with the aid of a Beckman Automatic Titrator. Selection of equilibrium pH values and ligand-metal ratios is dependent on the formation constants calculated for each system. Enzyme kinetic studies are used in an effort to determine the nature of the inhibition of acetylcholinesterase. It is found that (a) 1-1 ligand-copper chelate inhibit the acetylcholinesterase system, (b) free cupric ion may inhibit the enzyme system if concentrations greater than 1 x 10-6M are present in the equilibrium mixture, (c) coordination vacancies on the metal ion are necessary prerequisites for inhibition, (d) 1-1 ligandcopper chelates of 1,3 diaminopropanol inhibit the enzyme in an essentially "competitive" manner, (e) 1-1 ligand-copper chelates of 1 ,3 diaminopropane inhibit the enzyme in an essentially "noncompetitive" manner, (f) 1-1 ligand-copper chelates of hydroxy

The upper bound of enzyme concentration for accurately estimating the parameters in Michaelis-Menten (MM) equation is not completely determined and still under discussion, even though many researchers have investigated the equation’s validity for a long time. In the paper, we broadly investigated the correlation between the system of ordinary differential equations for monosubstrate irreversible enzyme reaction (HMM-system) and its derivative MM equation focusing on the relationship between initial enzyme concentration [E]0 and Michaelis constant Km by numerical simulation. According to the results, the initial reaction velocity v0 is still a function of initial substrate concentration [S]0 at [E]0

Phosphotransacetylase activity and the kinetic properties of the enzyme from intestinal sulfate-reducing bacteria Desulfovibrio piger and Desulfomicrobium sp. has never been well-characterized and has not been studied yet. In this paper, the specific activity of phosphotransacetylase and the kinetic properties of the enzyme in cell-free extracts of both D. piger Vib-7 and Desulfomicrobium sp. Rod-9 intestinal bacterial strains were presented at the first time. The microbiological, biochemical, biophysical and statistical methods in this work were used. The optimal temperature and pH for enzyme reaction was determined. Analysis of the kinetic properties of the studied enzyme was carried out. Initial (instantaneous) reaction velocity (V0), maximum amount of the product of reaction (Pmax), the reaction time (half saturation period, τ) and maximum velocity of the phosphotransacetylase reaction (Vmax) were defined. Michaelis constants (Km) of the enzyme reaction (3.36 ± 0.35 mM for D. piger Vib-7, 5.97 ± 0.62 mM for Desulfomicrobium sp. Rod-9) were calculated. The studies of the phosphotransacetylase in the process of dissimilatory sulfate reduction and kinetic properties of this enzyme in intestinal sulfate-reducing bacteria, their production of acetate in detail can be perspective for clarification of their etiological role in the development of the humans and animals bowel diseases. These studies might help in predicting the development of diseases of the gastrointestinal tract, by providing further details on the etiology of bowel diseases which are very important for the clinical diagnosis of these disease types.

Enzymatic reaction kinetics for sugar factory wastewater biological treatment was investigated. A laboratory scale completely mixed reactor was used for the study. By using the Runge-Kutta method and modified simple, estimation of kinetic parameters was conducted. The kinetic parameters rmax (the maximum reaction velocity) and Km (Michaelis constant) were found to be 0.7952×10 mg/min.L and 0.2519×103mg/L for COD, and 0.1359×10 mg/min.L and 0.2062×103 mg/L for NH3-N, respectively. These parameters could be utilized for the design of the three phase biological fluidized bed reactor for sugar factory wastewater treatment.

Enzymes bind to their substrates and transform them into products. A plot of the initial reaction velocity versus substrate concentration depicts a rectangular hyperbola. The reaction velocity (v) equals ( V max [A])/( K m + [A]) as described by the Michaelis-Menten equation where V max is the maximal velocity, [A] is the substrate concentration, and K m is the Michaelis constant, or the substrate concentration at half maximal velocity. Steady-state enzyme kinetics are used to determine the K m value for substrates, the V max value for enzymes, and the K i values for various inhibitors, including drugs.

The effects of bile salt cosurfactants on the lipase-catalyzed hydrolysis of p-nitrophenyl palmitate (p-npp) in AOT reversed micelles are described. Both an anionic bile salt, sodium taurocholate (NaTC), and a zwitterionic bile salt, 3-[(3-cholamidyl-propyl)dimethylammonio]-1-propanesulfonate (CHAPS), increase the reaction velocity of the hydrolysis reaction and the stability of the enzyme in the reversed micelles. CHAPS provides significantly larger increases in activity and stability than NaTC. Fluorescence studies indicate that addition of bile salt serves to immobilize the protein at the detergent layer and decrease the polarity experienced by the protein at the interface. Comparison of the results for lipase with previous results for other enzymes in the AOT/bile salt reversed micellar system lead us to conclude that the mechanisms by which bile salts affect enzymatic activity and stability in AOT reversed micelles vary from protein to protein. The effects, which generally are positive (increasing ac...

Propane and butane have very low reaction velocity and are abundant in the atmosphere of the Mexico City Metropolitan Zone (MCMZ). An important contribution of those alkane to ozone formation is observed. The present work studies to what extent the atmospheric concentrations and emissions of propane and butane contribute to the ozone formation. This evaluation was realized with the trajectory photochemical model, emissions and meteorological data of the MCMZ. Propane contributes to the maximum of ozone concentration with 0.013 ppm (7.78% of the total) and butane with 0.027 ppm (15.34% of the total). The total contribution of propane and butane to ozone formation can be split, respectively, in 0.001 ppm and 0.013 ppm of ozone produced by emission, and 0.012 ppm, and 0.014 ppm of ozone produced by atmospheric concentrations from the initial conditions. Propane and butane have a total contribution to a maximum of ozone concentration of 23.12%. The maximum concentration of propane has a decrement of 68.62% in the photochemical modeling.

Various parameters such as type and concentration of crosslinking agent, enzyme concentration, pH of the coupling medium and coupling time were optimized for the immobilization of urease on crosslinked poly(vinyl alcohol). Immobilized urease was thoroughly characterized for pH, temperature, solvents and storage stabilities and these properties were compared with the free enzyme properties. The Michaelis constant (Km) and maximum reaction velocity (Vmax) were calculated from Lineweaver-Burk plots for both free and immobilized enzyme systems. The energy of activation (Δ Ea) and the thermoinactivation constant (Ki) were calculated from the effect of temperature study. The urease-(PVA-F)-system shows good retention of activity even after 6–7 cycles.

The efficiency of penicillin G acylase catalyzed transformations is enhanced significantly with respect to reaction velocity, and in particular, stereoselectivity by appropriate “substrate tuning”, i.e. by the introduction of a nitrogen atom into the part of the substrates which is recognized by the enzyme.

Noninvasive visualization and investigation of interactions among proteins, biomolecules, and enzymes in living cells is an important goal for biologists, and fluorescence probes are powerful tools for this purpose. Because many target molecules are present in only trace amounts, high sensitivity is very important, and it is common to improve the sensitivity of fluorescence probes by focusing on high reaction velocity, K(d). (Gee, K. R.; Archer, E. A.; Lapham, L. A.; Leonard, M. E.; Zhou, Z.; Bingham, J.; Diwu, Z. Bioorg. Med. Chem. Lett. 2000, 10, 1515-1518.) So far, we have designed and synthesized various highly sensitive fluorescence probes based on the above concepts. (Gabe, Y.; Urano, Y.; Kikuchi, K.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2004, 126, 3357-3367. Komatsu, K.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2007, 129, 13447-13454.) Nevertheless, they were sometimes insufficiently sensitive to detect biomolecules in living cells, despite high chemical sensitivity in cuvette. In this report, we suggest a new approach to increase the sensitivity of fluorescence probes, focusing on their intracellular retention. Since calcein is well-retained, we investigated its structural, chemical, and optical characteristics and found that the iminodiacetic acid group (IAG) is a key structure for the intracellular retention. We next designed and synthesized novel fluorescence probes containing IAGs. They showed superior intracellular retention, making it possible to visualize low concentrations of target molecules that would be difficult to observe with conventional probes and permitting long-term observation in living cells. Improvement of intracellular retention of fluorescence probes holds great promise as a strategy for developing a wide range of highly sensitive probes for studies on various biological phenomena.

TiCl4−Lewis base (AcOEt, CH3NO2) complexes smoothly deprotected tert-butyldimethylsilyl (TBDMS) ethers. The reaction velocity with these complexes, which seemed less reactive due to the influence of Lewis bases, was considerably greater than that with TiCl4 alone. Selective desilylations between aliphatic and aromatic TBDMS ethers (1 and 5), between 1 and benzyl, allyl, tosyl, methoxyphenyl, and chloroacetyl ethers (13, 14, 15, 16, and 17), and between TBDMS and TBDPS ethers (18 and 19) were successfully performed. Desilylation of TBDMS-aldol, acyloin, and β-lactam analogues 9−12 proceeded smoothly due to anchimeric assistance by the neighboring carbonyl groups. The present method was successfully applied to the practical synthesis of 1β-methylcarbapenems 20a‘−f‘.

The kinetics of the phase I enzyme aspirin esterase were estimated in plasma from ten young volunteers, seven healthy elderly volunteers, and in ten frail elderly subjects. Maximal reaction velocity at enzyme saturation (vmax) was similar in young and healthy elderly volunteers, but was significantly reduced in the frail elderly. The Michaelis constant (Km) was similar in all three groups. The results suggest that impaired in vitro aspirin metabolism in frail elderly people is due to a reduction in the quantity of enzyme present, rather than a qualitative change in enzyme proteins.

Interactions between microwave energy (3 GHz) and the enzyme Lactate Dehydrogenase (LDH) have been analyzed by monitoring the enzymatic activity during irradiation in steady-state or dynamic conditions, by irradiating the sample with variable power levels (up to 6 W into the sample) and, finally, by knowing accurately the true specific absorption rate.No permanent or temporary changes can be induced when the energy absorption does not cause a temperature variation. For higher energy values, effects are purely thermal in nature. Furthermore the thermal activation of the reaction velocity, caused by microwave irradiation, is in itself sufficient to give a good fit with the experimental time evolution of the enzymatic reaction.

The chitin synthetase of Phycomyces blakesleeanus mycelium is a particulate enzyme sedimenting mostly at 1000xg. The activity in crude extracts or cellular fractions can be increased more than tenfold by mild trypsin treatment. Plotting the reaction velocity versus UDP-N-acetylglucosamine concentration yields a sigmoidal curve. N-acetylglucosamine, which greatly stimulates the enzyme, changes the kinetics to an almost normal hyperbolic relationship.

The rate of reaction of iron and hydrogen sulfide has been studied in an oxygen-free system in the presence of sodium chloride brine, in the presence and absence of an oil phase under a constant mixing rate at 40 G and 80 C. The experimental data are plotted according to a previously derived equation for the iron-acetic acid system, and are shown to be in agreement with that equation. Values of K, the overall reaction velocity, are obtained from the slopes. From the K values for three sets of experiments at 40 C and 80 C, values for the temperature coefficient are calculated, and are shown to correspond to the range of values expected for diffusional control. The presence of an oil phase seems to have little influence on the reaction rate. Another group of measurements on the iron-hydrogen sulfide system was carried out in the presence of corrosion inhibitors. While all the data of the inhibited reactions will show reasonable straight line plots, when plotted according to the diffusional equation, such plots have little meaning with the good inhibitors, because there is no essential change with time in the loss-in-weight values when they are used at 50 ppm. There seems to be some possibility that good inhibitors function by forming a particularly efficient non-permeable adsorbed film with iron sulfide corrosion products. 6.2.2

1. 1. An automated method was developed for the estimation of enzymatic activities based on the slope measurement of linear reaction curves. The procedure is applicable to most spectrophotometric assays, even when enzymatic reaction velocity is measured by means of a subsequent chemical reaction. 2. 2. The method was developed in the particular case of soluble starch hydrolysis by rat pancreatic amylase, estimated by the reduction of 3,5-dinitrosalicylic acid. Good kinetic graphs were obtained at a rate of 12 assays per hour for 1.8 min recording time.

The multi-phases parallel model for blend polymer solids was applied to express the mechanical properties of polymer film in the course of sorption of small molecules; the following relations were obtained.1) The following fundamental equations developed. With X: film thickness, x: distance from the film surface in the direction of the thickness, a: distance where the small molecules are diffused, C(x, t): concentration distribution at time t (mass/volume), q (t ): amount of absorbed small molecules at t (mass/volume), E: elastic modulus of polymer during the sorption process, EA(x, t): elastic modulus distribution, Ep: elastic modulus of pure polymer, F: function between EA and C at time t.2) When the relation between EA and C is linear, equation (9) is obtained by using the equations (3), (4) and (5).3) Assuming the three typical distributions of concentration and elastic modulus, the relations between E and q(t)/q(∞) were calculated for various relations between EA and C (see fig. 3 and fig. 4).4) The elastic modulus on the surface of film, EA(O, t) is given by following euqation. With ka: reaction velocity constant.5) Characteristic temperature dispersions of complex elastic modulus for polymer film during the sorption process were obtained by using the blend polymer theory (see fig. 6)

1. Assumed as monomolecular reaction, the velocity of the decom-position of potassium monochloracetate in aqueous solutions is measured, K_25°=0.837×10^-6 and K_30°=3.21×10^-62. The velocities of the following reactions are measured, CH2CLCOOK+KOH = CH2OHCOOK+KCL K_20°=0.227×10^-4, K_25°=0.521×10^-4 and K_30°=1.23×10^-4 CH2CLCOONa+NaOH=CH2OHCOONa+NaCl K_25°=0.381×10^-4, K_30°=0.704×10^-4 and K_35°=2.18×10^-4The velocity constant of potassium salt is larger than that of sodium salt. 3. The reaction velocity of potassium monochloracetate with potassium hydroxide is retarded by the presence of another salt (i.e. KNO3).

The influences of temperature and aldehyde concentration over the reaction velocity in acetalizing PVA fibre with acrolein and the reaction velocity depending upon the diffusion velocity of reagent into fibre were sought. The degrees of swelling, tenacities and elongations of the fibres were measured, and found that this kind of treatment is of no use for particular improvement of mechanical properties of the fibre.

The purification and properties of a Na+- and K+-activated ATPase from ox brain is described. The enzyme preparation is characterized by a high purity and a low content (< 1%) of Mg++-stimulated ATPase.

A method for the determination of enzyme kinetic constants Vm, Km, and Ki in a histochemical system has been devised. As a substitute for the reciprocal of the reaction velocity, the times necessary to reach a fixed amount of end product (the initial visible color) in a tissue site at various substrate concentrations are plotted, according to the method of Lineweaver and Burk, against the reciprocal of the substrate concentrations. The technique as applied to trypsin-like esterase and amidase activities in human mast cells indicates that a single enzyme or closely related enzymes in this site are responsible for the hydrolysis of both the amide and ester substrates and that typical trypsin substrates act as competitive inhibitors of their hydrolysis. Parallel biochemical studies were performed to evaluate the effect of certain aspects of the experimental histochemical method on a purified homospecific enzyme. The relative kinetic constants derived by the histochemical method afford a further means of characterizing enzymic activity in a histochemical system.

Die Synthese von Polyestern aus Dicarbonsaureanhydriden und cyclischen Diolkohlensaureestern erfordert Initiatoren. Sie wurde am Beispiel der Initiierung mit Alkalisalzen organischer und anorganischer Sauren untersucht. Die Zunahme der Reaktionsgeschwindigkeit mit der Polaritat des Reaktionsmediums, die gleichsinnige Anderung der Reaktionsgeschwindigkeit mit der Ionenleitfahigkeit der Initiatorsalze im Reaktionsmedium bei verschiedenen Konzentrationen, die Tatsache des Einbaus der Initiatoranionen in das Polyestermolekul und der Abbruch des Kettenwachstums durch Substanzen mit reaktionsfahigem Wasserstoff fuhren zu einem anionischen Reaktionsmechanismus. Initiators are necessary for the synthesis of polyesters from dicarboxylic acid anhydrides and cyclic carbonates of dihydric alcohols. This reaction has been studied as an example of initiation by alkalisalts of organic and inorganic acids. The increase of reaction velocity with the polarity of reaction medium, similar changes of reaction velocity with ionic conductivity of the initiator salts in reaction medium at different concentrations, the fact of incorporation of initiator anions in the polyester molecules and the termination of chain growth by substances having reactive hydrogen, indicate an anionic mechanism.

Es werden die spezifischen Geschwindigkeitskonstanten der alkalischen Solvolyse von Dimethyl-alkyl-silanen in Athanol/Wasser gemessen und mit fruheren Ergebnissen an Trialkyl-silanen verglichen. Die Alkyl-Gruppe beeinflust die Reaktionsgeschwindigkeit vermittels induktiver und sterischer Effekte. Specific rate constants of the alkaline solvolysis of dimethyl-alkyl silanes in ethanol water are determined and compared with former results about trialkyl silanes. The alkyl group influences the reaction velocity by inductive and steric effects.

Cellosolve acetate, methyl acetate, ethyl acetate, n-butyl acetate and iso-amyl acetate were investigated for alkali hydrolysis at 20 and 30°C, and for acid hydrolysis at 70°C.More positivity of the ester combination resulted higher reaction velocity in case of saponification reaction.In the case of acid catalized hydrolysis the velocity of reaction was very low, and cellosolve acetate did not react so higher than the methyl acetate. Cellosolve acetate had higher velocity of hydrolysis than other alkyl acetate in hydrophility.

Chromogenic substrates allow those interested in blood clotting and fibrinolytic enzymes to follow the activity of these enzymes continuously with the aid of a spectrophotometer. This is a major new tool in the study of these enzymes. Like any new tool, it takes some time to become acquainted with its possibilities and limitations so that we can use it optimally. This communication will stress some of the more obvious pitfalls and snags that stand in the way of an advantageous use of these substances. I will discuss the following main points

The reaction pathway catalysed by pyruvate carboxylase was re-examined by using two independent experimental approaches not previously applied to this enzyme. To avoid the variable stoicheiometry associated with oxaloacetate formation, the reaction rate was measured by following release of Pi. Initial velocities, when plotted as a function of varying concentrations of either MgATP2- or HCO3-, at fixed concentrations of pyruvate, gave in double-reciprocal-form families of straight intersecting lines. Further, when the reaction velocity was determined as a function of varying MgATP2- concentrations by using pyruvate, 3-fluoropyruvate and 2-oxobutyrate as alternative carboxyl-acceptor substrates, the slopes of the double-reciprocal plots were significantly different. Both results support a sequential reaction pathway.

Heparin is a substance extracted from animal tissues, formed by acidic carbohydrate long chains polymers (amino sugar containing sulfated mucopolysaccharides). It is used in therapy for its powerful anticoagulant activity (1). In fact the mucopolysaccharide achieves a dramatical increase of the reaction velocity between proteinases of the clotting system and at least two plasma proteinase inhibitors: antithrombin III (2,3) and heparin cofactor II more recently described (4,5).

When reaction velocity measurements of enzyme reactions are carried out with single beam, single monochromator spectrophotometers, stray light in the spectrophotometer can produce systematic errors in the apparent velocities when highly absorbing solutions (optical density >2.0) are used. These errors can give rise to spurious “inhibition” patterns of the steady state kinetics. Because of a suspected error of this kind, this laboratory has recently reinvestigated the kinetics of glucose 6-phosphate dehydrogenase from Escherichia coli and found that the reported noncompetitive inhibition of the enzyme by DPNH is explained more readily by an unnoticed effect of stray light on the apparent reaction velocity than by a true enzyme inhibition. Methods for estimating and correcting such errors in spectrophotometers are presented in detail.

The effects of volatile anaesthetics on bacterial luciferase were studied in vitro . It was shown that the concentration of anaesthetic required to inhibit the reaction velocity by 50% was similar to that required to reduce light output by 50% in vivo and this concentration was also in the clinical range for each agent. A kinetic response suggestive of competitive inhibition is occuring at the aldehyde binding site on the luciferase and it is postulated that this is related to the very hydrophobic nature of this site.

Resident mouse peritoneal macrophages contain a phospholipase C of high activity that is specific for phosphatidylinositol. The activity has a neutral pH optimum, is Ca2+-dependent and has a maximum reaction velocity of 525nmol/h per mg of protein. Certain phenothiazines are potent inhibitors of this activity.

Algebraic derivations demonstrate that if a multireactant enzyme system is poised at equilibrium and the concentration of one of the reactants is then changed by a small fraction, the resulting net reaction velocity is hyperbolically related to the fractional perturbation rather than the initial or final absolute concentration of that reactant. For small fractional perturbations the velocity is almost identical regardless which reactant is perturbed. Similar results are obtained even if the reaction system is already displaced by up to 30% from equilibrium at the time of the perturbation. These conclusions are independent of the relationships between the reactant concentrations and the kinetic constants for the enzyme. Thus under any near-equilibrium condition each of the reactants for a multireactant enzyme system shares almost equally in control of the net reaction velocity.

The inhibition of aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) by L-hydrazinosuccinate has been studied. The velocity of the enzyme reaction decreased with time when the reaction was initiated by the addition of enzyme to a mixture of the assay components and L-hydrazinosuccinate, while it increased slowly from a low level when a preincubated mixture of the enzyme and the inhibitor was added to the reaction mixture to initiate the reaction. Nearly 50% decrease in the initial reaction velocity was produced by a prolonged preincubation of the enzyme with the inhibitor, both at low concentrations of about 2 nM. These findings indicate that the inhibition is of the slow- and tight-binding type. The time-course of the reaction of the enzyme and the inhibitor, examined by the change in activity, was not in accord with single-step mechanisms, but rather appeared to follow biphasic kinetics. The inhibition could be fully reversed only in the presence of L-cysteine sulfinate or large excess of L-aspartate to convert the regenerated enzyme to its pyridoxamine form. The time-course of the reversal followed pseudo-first-order kinetics. Quantitative analysis of the experimental data has shown that the results are consistent with a mechanism of enzyme-inhibitor interaction which involves a reaction of two consecutive, reversible steps. The overall inhibition constant for L-hydrazinosuccinate was calculated to be approx. 0.2 nM.

Bilirubin was found to be a potent inhibitor of NAD+-linked isocitrate dehydrogenase (threo-ds-isocitrate:NAD+ oxidoreductase (decarboxylating), EC 1.1.1.41). It inhibited the enzyme by decreasing the affinity of enzyme for isocitrate, while the maximum reaction velocity was not affected. Bilirubin (less than 10−6 M) showed a detectable inhibition. On the other hand, neither the mitochondrial nor the cytoplasmic NADP+ enzymes (threo-ds-isocitrate:NADP+ oxidoreductase ((decarboxylating), EC 1.1.1.42) were affected by bilirubin.

Summary The first part of this lecture is devoted to some aspects of the study concerning the relationship between chemical structure and biological activity of lysozymes: role of the N -terminal sequences and of the tryptophan (modification by ozonization), histidine and cystine (heat stability) residues. In the second part are summarized some recent results obtained in the studies of goose and duck egg-white lysozymes and of human lysozymes (normal and from patients suffering from leukemia); the N -terminal sequences (53 amino acids) of two duck egg-white lysozymes are reported. Some observations on the mechanism of action of the lysozymes can be found in the conclusion (substrates, reaction velocity, specificity, transglycosylation reactions, behaviour in the presence of inhibitors).

The theory of the kinetics of enzyme cascades is developed. Two types of cascades are recognized, one in which the products are stable ( open cascades ) and another in which the products are broken down ( damped cascades ). It is shown that it is a characteristic of a cascade that the final product appears after a certain lag phase. After this lag phase, the velocity of product formation can be very rapid. It is shown that whereas open cascades will always show a complicated time–product relation, damped cascades can under certain circumstances resemble a simple enzymic reaction. Because the relation between the over-all reaction velocity in the extrinsic coagulation cascade and the concentration of any of the proenzymes in this cascade is a hyperbolic one, it is concluded that this cascade is of the damped type rather than the open type.

Semi-purified preparations of histidine decarboxylase from hamster placenta and from fetal rat tissues have been studied. The properties of the histamine-forming enzyme from hamster placenta are very similar to those of fetal rat histidine decarboxylase. Both enzymes can be extracted and purified by the same technique and their kinetic properties appear identical. With saturating concentrations of histidine the apparent maximal reaction velocity is around pH 5.5, but it could be established that the pH optimum of the reaction is inversely related to the substrate concentration. The apparent K m decreases with increasing pH. Enzymes with similar properties have been demonstrated in some other tissues of the rat and mouse; the apparent substrate specificity seems to justify the use of the name histidine decarboxylase to denote this enzyme, or group of enzymes.

1. 1. Some aspects of the kinetics of the CPK catalysed reaction PC + ADP ⇌ ATP + creatine and the effects of certain arsenical inhibitors have been studied. 2. 2. CPK is activated by Ca +2 , Mg +2 and Mn +2 , and the reaction velocity is greatest in the presence of the latter ion. 3. 3. The effects of lewisite and diphenylchloroarsine suggest that CPK is a “monothiol” enzyme.

Summary The monovalent cation requirements of formyltetrahydrofolate synthetase (formate:tetrahydrofolate ligase (ADP), EC 6.3.4.3) and the mechanisms by which di-and monovalent cations activate the enzyme were examined. The enzyme shows an absolute requirement for a monovalent cation. The relative effectiveness of the cations tested is NH 4 + >K + = Rb + >Cs + >Na + >Li + . It was found that the K m values of formate and tetrahydrofolate increased as the concentration of NH 4 + was lowered from 50 to 0.8 mM. The same change in NH 4 + had no effect on the maximum velocity of the reaction at infinite concentrations of either formate or tetrahydrofolate. On the other hand the maximum velocity at infinite ATP was influenced by NH 4 + concentrations. The results indicate that the enzyme binds with the monovalent cation before tetrahydrofolate and formate. The binding of ATP is, however, independent of NH 4 + . Spermine can replace NH 4 + to a certain degree but does not stimulate the reaction velocity in the presence of a saturating amount of NH 4 Cl. In addition, the results of kinetic data support the view that Mg 2+ activation of the enzyme results from complex formation between ATP and Mg 2+ to form the substrate, MgATP * .

The last decade has seen an exponential expansion of interest in conjugating multiple enzymes of cascades in close proximity to each other, with the overarching goal being to accelerate the overall reaction rate. However, some evidence has emerged that there is no effect of proximity channeling on the reaction velocity of the popular GOx-HRP cascade, particularly in the presence of a competing enzyme (catalase). Herein, we rationalize these experimental results quantitatively. We show that, in general, proximity channeling can enhance reaction velocity in the presence of competing enzymes, but in steady state a significant enhancement can only be achieved for diffusion-limited reactions or at high concentrations of competing enzymes. We provide simple equations to estimate the effect of channeling quantitatively and demonstrate that proximity can have a more pronounced effect under crowding conditions in vivo, particularly that crowding can enhance the overall rates of channeled cascade reactions.

By systematically varying the general assaying procedure, students can observe reaction velocity as a function of enzyme concentration, reaction velocity as a function of pH, enzyme activity as a function of temperature, and reaction velocity as a function of substrate concentration.