Enzyme velocity

The three most common types of inhibitors in enzymatic reactions are competitive, non-competitive, and uncompetitive. Competitive inliibition occurs when tlie substrate and inhibitor have similar molecules that compete for the identical site on the enzyme. Non-competitive inhibition results in enzymes containing at least two different types of sites. The inhibitor attaches to only one type of site and the substrate only to the other. Uncompetitive inhibition occurs when the inhibitor deactivates the enzyme substrate complex. The effect of an inhibitor is determined by measuring the enzyme velocity at various... 

Figure 11-9. Enzyme velocity versus substate concentration.

Subsequently Briggs and Haldane (1925) demonstrated that a similar treatment could be used to describe steady state enzyme velocity as a saturable function of substrate concentration ... 

Mutual exclusivity can also be tested for by the effects of combinations of two inhibitors on the activity of a target enzyme. The advantage of this approach is that it does not require any special labeling of either compound, and only catalytic quantities of enzyme are required for the studies. There are a number of graphical methods that can be used to determine the effects of inhibitor combinations on enzyme velocity (see Copeland, 2000). The most popular of these was introduced by Yonetani and Theorell (1964) and is based on the following reciprocal equation ... 

Here vy is the enzyme velocity in the presence of both compounds at concentrations [/] and [J], The term y is an interaction term that defines the degree to which binding of one compound perturbs the affinity of the enzyme for the second compound. 

If two compounds bind in a mutually exclusive fashion, then their effects on enzyme velocity are additive and the value of y is infinite (i.e., the combination term... 

Today, it is accepted that Langley and Ehrlich deserve comparable recognition for the introduction of the receptor concept. In the same years, biochemists studying the relationship between substrate concentration and enzyme velocity had also come to think that enzyme molecules must possess an active site that discriminates among various substrates and inhibitors. As often happens, different strands of evidence had converged to point to a single conclusion. 

There are a number of interchangeable words for velocity the change in substrate or product concentration per time rate just plain v (for velocity, often written in italics to convince you it s special) activity or the calculus equivalent, the first derivative of the product or substrate concentration with respect to time, d[P]/dt or —d[S]/dt (the minus means it s going away). Regardless of confusion, velocity (by any of its names) is just how fast you re going. Rather than miles per hour, enzyme velocity is measured in molar per minute (M/min) or more usually in micromolar per minute (p-M/min). 

Titrimetric analyses have established that Ki = I05-41 ooa M l and K2 = 10Z-34 0°3 M-1 (54, 58). With use of these values and of conservation equations for both Mg and PP, the respective concentrations of Mgz+, PP], MgPPf, and MgzPP- in any given assay mixture can be readily calculated. Enzymic hydrolysis (PPi 2 P,) was measured in each of a large number of solutions in which the concentrations of the various PPi species varied widely. The enzyme velocities so obtained were correlated with these concentrations and analyzed for mathematical fit to a number of possible kinetic models. [At lower pH additional ionic species are present, for example, HPP-- and MgHPP-". However, at pH 9.1 where enzymic activity is maximal (Section III,A), these protonated species are virtually nonexistent and can be neglected.]... 

Volume of first and second vessels Liquid volume Enzyme velocity constant Maximum enzyme velocity constant in unprotonated form Initial enzyme velocity constant Enzyme velocity constant based on unit volume of immobilised biocatalyst Maximum rate of reaction involving substance S Maximum rate of reaction involving substance P Specific rate of generation of biomass fraction Biomass concentration Initial or feed biomass concentration Average biomass concentration Concentration of prey Concentration of predator Biomass concentration at optimum dilution rate... 

Enzyme kinetics Km Michaelis-Menten constant is substrate concentration [S] that produces half maximum enzyme velocity v enzyme velocity, V maximum enzyme velocity... 

Figure 2. Effect of changing enzyme concentration on enzyme velocity. Velocity calculated according to Equation 3 with V ,ax = kcatlEnzyme] and K , = [S].

Note The enzyme of resistant insects is a little less efficient by having a slightly higher Km value. This indicates a somewhat less efficient enzyme, but the difference is so slight that it does not cause any reduced fitness for insects because the amount of and activity of acetylcholinesterase are almost always much higher than strictly necessary. LD50 = lethal dose in 50% of the population. Vm = maximum enzyme velocity. 

Figure 8.14 Plot of enzyme velocity, v, as a function of substrate concentrations, [Sj.

A) The enzyme velocity is at 2 the maximal rate when 100% of the enzyme molecules contain bound substrate. 

D) The enzyme velocity is at its maximal rate when all of the substrate molecules in solution are bound by the enzyme. 

The spectrophotometric data for the immobilized enzymes colloidal systems showed marked light scattering effects. The data were therefore subjected to a smoothing function by curve fitting a (best fit) fourth order polynomial to the data set from 18 to 80 seconds (the time period investing Vmax as determined from the data set of fresh (day 0) free enzyme activity). After curve fitting, the derivatives were calculated and enzyme velocity (VI8-80) was determined for each of the four systems (free enzyme, surface immobilized, inactivated surface immobilized, and immobilized supernatant) over the four time intervals (day 1, 3, 5 and 7). The data were then normalized to the activity of the free enzyme and plotted. 

Figure 2. Typical spectrophotometric data for the first 200 seconds. The nanocrystalline particulates induced marked light scattering which necessitated mathematical smoothing of the data before a slope (enzyme velocity) could be calculated.

Fig. 2. Reaction of 3 -p-fluorosulfonylbenzoyladenosine with bovine liver glutamate dehydrogenase. Glutamate dehydrogenase (021 mg/ml) was incubated with 3 -FSBA (0.496 mil/) at 24° in 0.01 M sodium barbital buffer (pH 8) containing 0.43 M KCl and 5% ethanol. At each indicated time, an aliquot was withdrawn, diluted 20-fold with Tris-0.1 M acetate buffer (pH 8) at 0°, and assayed (A) in the absence and (B) in the presence of 100 yM ADP. Inset Determination of the pseudo first-order rate constant from the decrease in activation by ADP. (Ft and Fo are the enzymic velocities measured in the presence of ADP and the given and zero time, respectively, and F > is the constant velocity at the end of the reaction. The pseudo first-order rate constant calculated is 0D351 min. ) Data are taken from P. K. Pal, W. J. Wechter, and R. F. Colman, Biochemistry 14, 707 (1975).

Monod kinetic parameters obtained for the reduction of uranyl and chromate (Table 6-3), using lactate as the electron donor, provide maximum enzyme velocities (Vmax) that are surprising similar (i) to maximum rates for other experimental conditions noted in Table 6-1 and 6-2, and (ii) between different organisms and different electron acceptors—Vn,gx differs by less than a factor of four for different organisms and for different TEAs (Gorby, 2000, personal communication). 

The overall enzyme velocity is expressed through probabdities ... 

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