It is the substrate concentration needed to achieve a half-maximum enzyme velocity. How does pH affect enzyme activity? Changing the pH outside of this range will slow enzyme activity. Extreme pH values can cause enzymes to denature. Enzyme concentration: Increasing enzyme concentration will speed up the reaction, as long as there is substrate available to bind to. Is kcat the same as Vmax? Because the concentration of enzyme is taken into account in this equation, Kcat does NOT vary with the amount of enzyme used and is therefore a constant for an enzyme.
Kcat is equal to the number of molecules of product made per enzyme per unit time. What is Km value? The Michaelis constant KM is defined as the substrate concentration at which the reaction rate is half of its maximal value or in other words it defines the substrate concentration at which half of the active sites are occupied. Can km be negative?
If you follow the disappearance of something, the velocity should be "negative" and hence you need to invert it to get a positive reaction velocity. Only when the reaction rate is positive will you find both Michaelis-Menten parameters to be positive.
Also, make sure your reaction rate is faster as [S] increases. Why is Lineweaver Burk plot more accurate? It is also more robust against error-prone data than the Lineweaver—Burk plot, particularly because it gives equal weight to data points in any range of substrate concentration or reaction rate the Lineweaver—Burk plot unevenly weights such points.
What happens when you increase the amount of substrate? Initially, an increase in substrate concentration leads to an increase in the rate of an enzyme-catalyzed reaction. As the enzyme molecules become saturated with substrate, this increase in reaction rate levels off. In practice, it is usual to use a concentration of substrate about 10 - fold higher than the Km in order to determine the activity of an enzyme in a sample.
If an enzyme is to be used to determine the concentration of substrate in a sample e. Km and Vmax are determined by incubating the enzyme with varying concentrations of substrate; the results can be plotted as a graph of rate of reaction v against concentration of substrate [S], and will normally yield a hyperbolic curve, as shown in the graphs above. The relationship is defined by the Michaelis-Menten equation:. I t is difficult to fit the best hyperbola through the experimental points, and difficult to determine Vmax with any precision by estimating the limit of the hyperbola at infinite substrate concentration.
A number of ways of re-arranging the Michaelis-Menten equation have been devised to obtain linear relationships which permit more precise fitting to the experimental points, and estimation of the values of Km and Vmax. There are advantages and disadvantages associated with all three main methods of linearising the data. The Lineweaver-Burk double reciprocal plot rearranges the Michaelis-Menten equation as:.
This is the most widely used method of linearising the data, and generally gives the best precision for estimates of Km and Vmax. These are the points at which the precision of determining the rate of reaction is lowest, because the smallest amount of product has been formed.
The Eadie-Hofstee plot rearranges the Michaelis-Menten equation as:. This will enable you to plot a graph of Velocity of reaction absorbance units per sec against Substrate concentration M. From the graph find the maximum velocity and half it i. Draw a horizontal line from this point till you find the point on the graph that corresponds to it and read off the substrate concentration at that point. This will give the value of Km. Sign up now.
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