Determination of Vmax & Km

These parameters can be derived from experiments, in which rates of enzyme reaction are determined at different substrate concentrations, provided that the enzyme obeys Michaelis-Menten rules. The values for Vmax and Km could be read off a hyperbolic plot of initial velocity versus [S], but in practice it is difficult to read accurately from a curved line. Fortunately the M-M equation is conveniently transformed into an equation that gives a straight line plot by taking reciprocals of both sides of M-M equation.

Thus a plot of 1/vo versus 1/[S] yields a straight line with an intercept on the y-axis of 1/Vmax, a slope = Km/Vmax, and intercept on the negative side of the x-axis of -1/Km. This double reciprocal plot is called a Lineweaver-Burke plot.

Units

The units of Km are those of concentration i.e. mM, mM or Km is the concentration of substrate at which half maximal velocity is observed. Vmax can be expressed in a variety of units depending on what information is available. If the enzyme preparation is not pure the units could be mmoles product formed/min or if pure enzyme used mmoles min/mg (enzyme). Recently an SI unit has been introduced called the katal which is amount of enzyme which catalyses production of 1 mole of product per second.

Significance of Km & Vmax kinetic parameters

Km values range widely but most lie between 0.1 and 0.000001 M. The Km for an enzyme depends on the particular substrate and on conditions of assay such as temp. & pH. Km represents the strength of binding or affinity of the substrate for the enzyme. Tightly bound substrates have a low Km e.g. mM and loosely bound substrates have a high Km e.g. mM. Also the Km is that concentration of substrate at which half the active sites of the enzyme are filled.

The maximal rate, Vmax reveals the turnover No. of an enzyme i.e. the number of substrate molecules being catalysed per second. This varies considerably from 10 in the case of lysozyme to 600,000 in the case of carbonic anhydrase.

For more detail on this topic see the text book pages 166-167

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