Enzyme kinetics units

The results from Basic Protocol 1 are expected to be consistent with traditional initial velocity assumptions for enzyme kinetics (unit cl /). The assay, as presented, includes four time points (along with a zero-time value) in order to establish the relationship between reaction time and product formed. Representative data, demonstrating the hyperbolic nature of this relationship, are presented in Figure C1.2.3. In this case, only the initial time points at the lowest enzyme concentration are consistent with the linear initial velocity assumption. If... 

Carrier-mediated transport is linear with mucosal solute concentration until this concentration exceeds the number of available carriers. At this point the maximal solute flux (7max) is independent of further increases in mucosal solute concentration. In the linear range of solute flux versus mucosal concentration (C), the proportionality constant is the ratio of / to the solute-carrier affinity constant (Km). This description of Michaelis-Menten kinetics is directly analogous to time changes in mass per unit volume (velocity of concentration change) found in enzyme kinetics, while here the appropriate description is the time change in solute mass per unit surface area of membrane supporting the carrier. 

We point out that in enzyme kinetics TON is understood as TOF It is also sometimes called the turnover number, because it is a reciprocal time and defines the number of catalytic cycles (or turnovers ) that the enzyme can undergo in unit time, or the number of molecules of substrate that one molecule of enzyme can convert into products in one unit of time. Quotation from [23]. 

This section mainly builds upon classic biochemistry to define the essential building blocks of metabolic networks and to describe their interactions in terms of enzyme-kinetic rate equations. Following the rationale described in the previous section, the construction of a model is the organization of the individual rate equations into a coherent whole the dynamic system that describes the time-dependent behavior of each metabolite. We proceed according to the scheme suggested by Wiechert and Takors [97], namely, (i) to define the elementary units of the system (Section III. A) (ii) to characterize the connectivity and interactions between the units, as given by the stoichiometry and regulatory interactions (Sections in.B and II1.C) and (iii) to express each interaction quantitatively by... 

Enzyme Nomenclature. Recommendations (1992) Academic Press, New York Nomenclature of multiple forms of enzymes J. Biol Chem. (1977) 252, 5939-5941 Catalytic activity Units of enzyme activity Eur. J. Biochem. (1979) 97, 319-320 Symbolism and terminology in enzyme kinetics Eur. J. Biochem. (1982) 128, 281-291... 

Concentration is the most common means for describing the composition of a solution in biochemistry. Enzyme kinetic expressions are typically expressed in these concentration units. Unless otherwise noted, this is the method used throughout this text. Nevertheless, other methods for describing compositions are utilized. For example, mole fractions are often used in Job plots. Gases in solution are commonly measured in terms of partial pressures. Below is a brief description of a few of these other conventions or methods. 

If you have a class with biochemists, clearly the area of enzyme kinetics is practically mandatory. If biologists are mixed in with the biochemists, osmotic pressure is an important concept to cover carefully and a concept typically not well covered in general chemistry and in most physical chemistry texts or classes. A quick example what is a 2 Osmolar solution of sodium chloride Such concentration units are used when dispensing various saline solutions in hospitals. What is the origin of the unit A 1 M NaCl solution dissociates into two ions that would double the osmotic pressure of a non dissociating solute. Thus, the 1 M solution of NaCl becomes a 2 Osmolar solution. Other examples abound - the bursting pressure of a cell relates to the osmotic pressure of the serum in which the cell finds itself. 

The presence or absence of an enzyme is typically determined by observing the rate of the reaction(s) it catalyzes. Quantitative enzyme assays are designed to measure either the total amount of a particular enzyme (or class of enzymes) in units of moles or, more commonly, the catalytic activity associated with a particular enzyme. The two types of assays differ in that those in the latter category measure only active enzyme. The assays contained in this section are concerned primarily with the measurement of catalytic activity, or active enzyme. The assays are based on kinetic experiments, as activities are calculated from measured reaction rates under defined conditions. The basic Premise for these assays is that the amount of enzyme in a reaction mi xture can be determined from the rate at which the enzyme-catalyzed reaction occurs. 

IUPAC and IUB (1972) Enzyme Nomenclature Recommendations of the Commission on Biochemical Nomenclature on the Nomenclature and Classification of Enzymes together with their Units and the Symbols of Enzyme Kinetics. Elsevier, Amsterdam. 

The rate constant expresses the proportionality between the rate of formation of B and the molar concentration of A and is characteristic of a particular reaction. The units of k depend on the order of the reaction. For zero order, they are moles liter" time" (time is frequently given in seconds). For first order, they are time" , and for second order, liters moles" time" , etc. The units of k are whatever is needed for zero order reaction, [B] changes at a constant rate independent of the concentration of reactants, which is especially important in enzyme kinetics. A plot of [B] versus t for such a reaction is a straight line. A somewhat more complicated example is... 

APases hydrolyze numerous phosphate esters, such as those of primary and secondary alcohols, phenols and amines (Levine, 1974). One unit of activity of APase corresponds to the hydrolysis of 1.0 pmole of p-nitrophenyl phosphate (p-NPP) per min (in 100 mM glycine, 1 mM ZnCb, 1 mM MgCl2 and 6 mM p-NPP, pH 10.4 or in 1 M diethanolamine, 0.5 mM MgCU and 15 mM p-NPP, pH 9.8). The bovine enzyme generally has a specific activity of 1000 and 2000 U/mg in these two buffers, respectively, at 37°C. At 25°C, activity is reduced to about half. This demonstrates that buffers may have a marked influence on the enzymatic activity of APases which explains the great differences in activity given for commercial preparations. Assays with p-NPP above 30°C suffer from the spontaneous hydrolysis of this substrate, with serious consequences for the enzyme kinetics (see below). The bacterial enzyme has lower activity than the bovine intestinal enzyme. 

CST reactors have been more widely adopted, partly due to the possibility of concentration polarization control, and partly due to the easy modeling of enzyme kinetic behavior. In the literature, comprehensive mathematical descriptions of the kinetic behavior of enzymes located in CSTR UF units are reported 9 1-0 as far as low-molecular-weight substrates are concerned. 

Fig. 9 NMR spectral changes revealed by a 5 mm solution of hyperpolarized choline upon undergoing phosphorylation by 0.5 units of choline kinase, (a) Emergence of the new phosphocholine resonance shown by directly detected single-pulse N NMR spectroscopy experiments, (b) Emergence of the H NMR resonance associated with the methylenes in the C2-position of phosphocholine, (c) Comparison between the expected enzyme kinetics of kinase with results afforded by the N- ( ) and H-detected ( ) hyperpolarized experiments, as derived from the relative peak ratios of the NMR peaks in (a) and (b). The straight line illustrates the best fit of the combined set of data points, and corresponds to an initial phosphorylation rate of 0.3 mM min under these conditions. Reproduced with permission from [55]...

According to the SI system, catalytic activity is defined by the katal (1 kat = 1 mol s of substrate transformed). Since its magnitude is far too big for practical application, it has not been widely accepted. The transformation of one mole of an organic compound within one second resembles an industrial-scale reaction and is thus not suited to describe enzyme kinetics. As a consequence, a more appropriate standard - The International Unit (1 I.U. = 1 pmol of substrate transformed per min) - has been defined. Unfortunately, other units such as nmol/min or nmol/hour are also common, mainly to make the numbers of low catalytic activity look bigger. After all, it should be kept in mind that the activities using nonnatural substrates are often significantly below the values which were determined for natural substrates. 

Deming and Pardue studied the kinetics for the hydrolysis of p-nitrophenyl phosphate by the enzyme alkaline phosphatase. The progress of the reaction was monitored by measuring the absorbance due to p-nitrophenol, which is one of the products of the reaction. A plot of the rate of the reaction (with units of pmol mL s ) versus the volume, V, (in milliliters) of a serum calibration standard containing the enzyme yielded a straight line with the following equation... 

In zero-orrler kinetics, a constant amount of a chemical compound is excreted per unit of rime. In most cases, this phenomenon is caused by the saturation of a rate-limiting enzyme, and the enzyme commonly functions at its maximal rate, i.e., a constant amount of a chemical compound is metabolized per unit time. A good example is ethyl alcohol alcohol dehydrogenase becomes saturated at relatively low concentrations. Because of this saturation, ethyl alcohol is eliminated at a constant rate about 7 g/h. However, rhe reason is not always an enzyme anv... 

Fibrinolytics. Figure 3 Plasminogen activation (a) Kinetics of plasminogen activation by uPA (urokinase-type) and tPA (tissue-type) plasminogen activators. Effect of fibrin (b) Ternary complex formation between enzyme (tPA), substrate (Pg) and cofactor (F) Abbreviations plasmin (P), fibrin (F), plasminogen (Pg). Plasmin, formed in time, is expressed in arbitrary units. 

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