General assumptions and guidelines in using approaches to Michaelis-Menten equation derivations: Rapid equilibrium approach: 1.

Initial rate data is measured as reaction velocities, v vo 2. The formation of the ES complex is much faster than its breakdown in forming P
Km k2 E S ES E P such that k 1 k 2 therefore, K m

k 1 k 2 k 1 which is a k1 k1

dissociation constant, or this time, Michaelis-Menten constant. 3. The concentration of the [ S o ] [ Eo ] (in 5 to 6 orders of magnitude difference) and substrate concentration will always be very high relative to the enzyme. If initial reaction velocities are measured, formation of ES complex starts from zero until it reaches an amount where it will be broken down to P 4. The conversion of ES complex to product from the rate-limiting step or rate-determining step
k2 E P is irreversible from #1 and #3 assumption where v k 2 .[ES ] (RDS) ES

d [ P] dt

5. It is usually appropriate in in-vitro enzymatic batch reactions. 6. It is easier to use in deriving complex enzyme reaction mechanisms.

Quasi Steady-state approach: (quasi = resembles, or almost same as) (formulated by Briggs and Haldane) 1. Initial rate data cannot be assumed since at the start of the reaction in the microseconds, ES complex is formed from 0, increases and starts to decline upon formation of P (see graph below). From the plot below showing relative concentrations of S, P, ES and E with time, they are not (except E) in steady-state where dC/dt is zero. But can be assumed to be constant especially if

d[S ] d [ ES] and not [ S o ] [ Eo ] dt dt

2. Also, it can be deduced that in the usual catalytic multiple reactions, the intermediates and complexes are difficult to measure since they are converted as fast as they are formed (no accumulation) that is why it is reasonable to assume,

d [ ES ] 0 . That is also because the product dt

is used in a series of reactions such as in biochemical pathways such that complexes and intermediates are consumed in another reaction fast as soon as it is formed so no accumulation happens. 3. While some have stated that

d [ ES ] 0 is because of the irreversibility in forming P, it is dt

k 1 k 2 k 1 and so k1 k1

irreversible since it will be used in a series reaction. 4. Rate contants k 1 has the same order of magnitude as k 2 therefore, K m

Km
5.

k 1 k 2 becomes the Michaelis-Menten constant k1

d [ P] is also assumed since the RDS is the breakdown of ES complex to P. But if dt d [ P] P is used as a substrate for subsequent reactions, we can therefore say that v k 2 .[ES ] . dt v k 2 .[ES ]
P does not accumulate in the same manner as ES. Quasi steady-state approximation is valid in this case, in a continuous enzymatic reaction.

6. It is more appropriate for in-vivo reactions involving enzymes where continuous biosynthesis occurs and where intermediates do not accumulate. 7. This assumption is more valid in most enzyme activities.

Engr. Aki Espaldon ChE513: Biochemical Engineering University of Santo Tomas