Substrate concentration:

If there are enzyme molecules with vacant active sites, an increase in substrate concentration will increase the rate of reaction. If the enzyme concentration is kept constant and the amount of substrate is increased, a point is reached where any further increase in substrate does not increase the rate of reaction anymore. When all the active sites of the enzymes are occupied (at high substrate concentration), any more substrate molecules do not find free active sites. This state is called saturation of active sites and the reaction rate does not increase.