Substrate the lower the substrate concentration range

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substrate, the lower the substrate concentration range in which the enzyme is effective. As we will soon see, enzyme activity in the cell can be modulated by regulatory molecules that bind to the enzyme and alter the K m for a particular sub- strate. K m values for several enzyme-substrate combinations are given in Table 6-2 and, as you can see, can vary over several orders of magnitude. Enzyme Name Substrate K m ( M ) k cat (sec uni2212.bold 1 ) Acetylcholinesterase Acetylcholine 9 uni00D7 10 - 5 1.4 uni00D7 10 4 Carbonic anhydrase CO 2 1 uni00D7 10 - 2 1 uni00D7 10 6 Fumarase Fumarate 5 uni00D7 10 - 6 8 uni00D7 10 2 Triose phosphate isomerase Glyceraldehyde-3-phosphate 5 uni00D7 10 - 4 4.3 uni00D7 10 3 b -lactamase Benzylpenicillin 2 uni00D7 10 - 5 2 uni00D7 10 3 K m and k cat Values for Some Enzymes Table 6-2 Figure 6-9 The Linear Relationship Between V max and Enzyme Concentration. The linear increase in reaction veloc- ity with enzyme concentration provides the basis for determining enzyme concentrations experimentally. V max V max = k 3 [E] Enzyme concentration [E]
141 Chapter 6 | Enzymes: The Catalysts of Life Figure 6-10 The Lineweaver–Burk Double-Reciprocal Plot. The reciprocal of the initial velocity, 1/ v , is plotted as a function of the reciprocal of the substrate concentration, 1/[S]. K m can be calcu- lated from the x -intercept and V max from the y -intercept. Slope = K m / V max y -intercept = x -intercept = 1/ v 1/[S] 1 K m V max V max v 1 K m 1 V max 1 1 [S] = + - Equation 6-12 on page 140 is known as the Lineweaver– Burk equation. When it is plotted as 1/ v versus 1/[S], as in Figure 6-10 , the resulting double-reciprocal plot is linear in the general algebraic form y uni003D mx uni002B b , where m is the slope and b is the y -intercept. Therefore, it has a slope ( m ) of K m / V max , a y -intercept ( b ) of 1/ V max , and an x -intercept ( y uni003D 0) of - 1/ K m . (You should be able to convince yourself of these intercept values by setting first 1/[S] and then 1/ v equal to zero in Equation 6-12 on page 140 and solving for the other value.) Therefore, once the double-reciprocal plot has been constructed, V max can be determined directly from the reciprocal of the y -intercept and K m from the negative recip- rocal of the x -intercept. Furthermore, the slope can be used to check both values. Thus, the Lineweaver–Burk plot is useful experimentally because it allows us to determine the parameters V max and K m without the complication of a hyperbolic shape. ( Key Technique, page 142 , shows an experimental set-up for determining these kinetic parameters for hexokinase, the en- zyme catalyzing the phosphorylation of glucose on carbon atom 6 to begin the degradation of glucose in the glycolytic pathway.) glucose uni002B ATP ++ ¡ hexokinase glucose @ 6 @ phosphate uni002B ADP (6-13)
PROBLEM: Hexokinase is an important enzyme in energy metabolism because it catalyzes the first step in the exothermic degradation of glucose. Understanding the kinetics of this reac- tion will help us to better understand its role in energy produc- tion in the cell. But how do scientists experimentally determine the kinetic parameters of K m and V max for an enzyme such as this?

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