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The active site is the special place, cavity, crevice, chasm, cleft, or hole that binds and then magically transforms the substrate to the product The kinetic behavior of enzymes is a direct consequence of the protein s having a limited number (often 1) of specific active sites
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mmol/mL and mol/ L are both the same as mol/L or M Other useful realizations include mM mol/mL
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Most of enzyme kinetics (and mechanism) revolves around the active site As we ll see later, saturation kinetics is one of the direct consequences of an active site
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An assay is the act of measuring how fast a given (or unknown) amount of enzyme will convert substrate to product the act of measuring a velocity
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How fast a given amount of substrate is converted to a product depends on how much enzyme is present By measuring how much product is formed in a given time, the amount of enzyme present can be determined An assay requires that you have some way to determine the concentration of product or substrate at a given time after starting the reaction If the product and substrate have different UV or visible spectra, fluorescence spectra, and so forth, the progress of the reaction can be followed by measuring the change in the spectrum with time If there are no convenient spectral changes, physical separation of substrate and product may be necessary For example, with a radioactive substrate, the appearance of radioactivity in the product can be used to follow product formation
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Velocity rate, v, activity, d[P]/dt, d[S]/dt is how fast an enzyme converts substrate to product, the amount of substrate consumed, or product formed per unit time Units are micromoles per minute ( mol/min) units
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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 ( M/min)
Enzyme Kinetics
Figure 8-1 shows what happens when enzyme is added to a solution of substrate In the absence of enzyme, product appearance is slow, and there is only a small change in product concentration with time (low rate) After enzyme is added, the substrate is converted to product at a much faster rate To measure velocity, you have to actually measure two things: product (or substrate) concentration and time You need a device to measure concentration and a clock Velocity is the slope of a plot of product (or substrate) concentration (or amount) against time Velocity can be expressed in a number of different units The most common is micromolar per minute ( M/min); however, because the velocity depends on the amount of enzyme used in the assay, the velocity is often normalized for the amount of enzyme present by expressing the activity in units of micromoles per minute per milligram of enzyme [ mol/(min mg)] This is called a specific activity You may be wondering (or not) where the volume went after all, product concentration is measured in molar units (M; mol/L) Well, it s really still there, but it
[Product] ( mol/mL)
velocity = slope
time (min)
velocity =
Figure 8-1
[Product] time
mLmin
The VELOCITY of product formation (or substrate disappearance) is defined as the change in product concentration per unit time It is the slope of a plot of product concentration against time The velocity of product formation is the same as the velocity of substrate disappearance (except that substrate goes away, whereas product is formed)
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