Elementary plant Biochemistry and Biotechnology (2+1)

1.Substrate concentration

Substrate concentration

•Keeping the factors such as pH, temperature and enzyme concentration at optimum levels, if the substrate concentration is increased, the velocity of the reaction recorded a rectangular hyperbola.
•At very low substrate concentration the initial reaction velocity (v) is nearly proportional to the substrate concentration (first order kinetics).
•However, if the substrate concentration is increased the rate of increase slows down (mixed order kinetics).
•With a further increase in the substrate concentration the reaction rate approaches a constant (zero order-reaction where velocity is independent of substrate concentration).
•At initial point, eventhough the substrate molecules are present in excess than enzyme on molar basis, not all the enzyme molecules present combine with the substrate.
•Hence, increasing the substrate concentration will increase the amount of enzyme associated with substrate as ES and thus v will depend on [S].
•At Vmax, all the enzyme molecules are saturated with substrate molecules so that further increase in [S] cannot result in increased reaction rate.
•Michaelis-Menten derived an equation to explain this type of behaviour.
Vmax [S]
v = -------------
Km + [S]
[S] = Substrate concentration Vmax = Maximum velocity
v = Velocity of the reaction
At half maximal velocity [S] = Km
i.e Vmax Vmax [S]
-------- = -------------
2 Km+[S]

Km + [S] Vmax [S]
----------- = ----------
2 Vmax
Km + [S] = 2 [S]
Km = 2 [S] – [S] = [S]
Hence, Michaelis - Menten constant, Km, is defined as the substrate concentration at half maximal velocity and is expressed as mole per litre.
•The Michaelis-Menten equation can be algebraically transformed into more useful way to plot the experimental data.
•Lineweaver and Burk have taken the reciprocal of both [S] and v of the Michaelis-Menten equation to give
1 Km 1 1
--- = ------ ------ + -------
v Vmax [S] Vmax
•A plot of 1/v versus 1/ [S] (the double reciprocal) yields a straight line.
•This line intercept X-axis at -1/Km and Y-axis at 1/Vmax.
•The slope of the line is Km/Vmax.
•The Lineweaver-Burk plot has the great advantage of allowing more accurate determination of Vmax and Km

Significance of Km
i. Km value may vary with substrate.
ii. An enzyme whose Km is very low will have a high degree of affinity for its
substrate