All the following assumptions apply to Michaelis-Menten kinetic analyses of enzyme action EXCEPT:
1. the total enzyme concentration studied at each substrate concentration is fixed in analysis of enzyme kinetics.
2. formation of enzyme-substrate complex does not appreciably decrease the concentration of substrate.
3. Km decreases with competitive inhibition.
4. maximal velocity is reached when the enzyme-substrate complex is equal to the total concentration of enzyme present.