ENZYME KINETICS

Michael-Menten Approach

Enzymatic reaction

Simplest enzymatic reaction 1st order (1) Real enzyme mechanism more complexes; never proceed through just 1 ES complex. Involved many steps pathway The slowest one determines the rate of overall reaction How the reaction rate is affected by reaction conditions (S,P,E) important, to understand the effectiveness & characteristics of enzyme reaction

The reaction will start at a high rate and slow down over time for several reasons: The substrate will be used up and the reaction rate will slow down as each enzyme molecule spends more time diffusing through the solution before it collides with a new substrate molecule. As the reaction proceeds, product will accumulate which may tend to inhibit the enzyme. The enzyme molecules may gradually lose activity owing to random denaturation.

Graph
Plotting r0 at different [S] & [E]: r is proportional to [S] (1st-order reaction) when the [S] is in the low range

[S] very low a straight line graph when [S] is low, [S] the limiting factor; an increase [S] produces a proportional increase in the rate

graph will curve at higher [S] & will level off at very high [S] The rate does not depend on the [S] when the [S] is high Reaction rate changes gradually from first order to zero order as [S] is increased. [S] increases, [E] the limiting factor a further increase in substrate produces a less than-proportional increase in reaction rate.

the enzyme becomes "saturated" and the reaction rate reaches a constant value doesn't increase significantly as yet more substrate is added.

ENZYME KINETICS

deals with the rate of enzyme reaction Rate (r), activity, dP/dt, (-dS/dt) how fast an enzyme catalyses S to P, the amount of S consumed, or P formed per unit time Rate ? need S or P, & t how reaction is affected by various chemical and physical conditions The rate equations calculating reaction time, yields For designing bioreactor and optimum economic condition

Factors affecting the rate of enzyme reaction

[S] the more, the quicker the enzyme molecules collide and bind with them). expressed in the unit of molarity, M T As the T rises, molecular motion speed up collisions between enzyme and substrate. Finite enzymes are proteins, have an upper limit beyond which the enzyme becomes denatured and ineffective. inhibitors a. Competitive inhibitors b. Noncompetitive inhibitors pH The conformation of a protein is influenced by pH and as enzyme activity is crucially dependent on its conformation, its activity is likewise affected.

Rate vs [S]
[S] low they are all bound to the enzyme molecules there are more substrate molecules some of them are free in solution, not bound to an enzyme molecule. [S] high all the enzyme molecules have a bound substrate. The number of enzyme molecules with bound substrate is an indication of the reaction rate those enzymes "act" and convert the substrate to product. The purple highlight indicates the region of the graph that corresponds to illustration in the left.

Henry (1902) observed this & proposed: (2) rmax & Km kinetic parameters, experimentally determined Brown (1902) E forms a complex with S. The complex then breaks down to the products, regenerates free enzymes (3) (4)

Assumptions: 1. The total enzyme concentration constant during the reaction; [Eo] = [ES]+[E] 2. The enzyme is very small amount compared to the amount of substrate the formation of the complex does not significantly deplete the substrate. 3. [P] is so low that product inhibition may be considered negligible

MICHAEL-MENTEN APPROACH
Assumption for MM approach: most enzymes has a fairly simple math shape a hyperbola product release step (4) is much slower than reversible reaction (3) ES complex has a weak interaction fast reaction; much faster step than product release step which involved chemical changes the slowest step determine the rate, the other is at equilibrium eventhough enzyme is soluble in water, enzyme molecules have large & complicated 3D structure

If slower reaction (4) determines overall reaction rate of P formation & S consumption is proportional to CES: (5)

Assumption MM no.3 CES =f(Cs, CE): (6) the rate of reaction can be expressed as a function of Cs and CE, CE difficult to be determined Total enzyme contents: (7)

Substitution (6) to (5) for CE & rearrange CES: (8)

Final rate eq: (9)

Know as MM eq identical to (2) Assumption: 1) only one intermediate state (ES complex), 2) dealing with initial rate (not necessary to consider back reaction) Km, Cs same unit (M or mol/L) Rmax is proportional to the CE0 rmax=k3CE0;difficulty to express CE0 in M unit

From MM eq

Initial [S] increase reaction goes faster; enzymes easier to find S All active sites occupied with bound S or P saturated [S]<<<[Km] double const, double rate rmax rate approached at saturated [S]; rate does not depend on [S] Km: [S] required to produce rate that is one-half of rmax Km >> the weaker the interaction between enzyme & S

[S]=Km rate=one-half of rmax [S]<<Km rate depends linearly on [S]; 2x[S] 2x rate [S]>>Km dependence of rate on [S] approach a max horizontal line/independent; rmax zero order kinetic

Theory shows that KM is approximately equal to the dissociation constant for the enzyme and the substrate, provided that the enzyme-substrate complex reverts to enzyme and substrate much more often than it goes on to generate product Enzymes have higher rates of reaction the substrate concentration is greater than KM. All enzyme-catalyzed reactions are reversible and if the concentration of product is high, the reverse reaction will compete with the forward reaction for enzyme. The rmax and KM values for the reverse reaction are usually different from those of the forward reaction.

Alternate Forms of the MM eq.

Where does KM & rmax come in? Estimate Km & rmax asymptote Km=f ([S]) Km low corresponds to tight binding of enzyme to substrate and vice versa KM is large the rate at low [S] is relatively low. KM is small the rate at low [S] is relatively high The curve of r vs [S] rise quickly if KM is low and the curve will soon approach Vmax

if KM is high, the curve will appear to spread out toward the right, approaching Vmax only at high substrate concentration. Km = rmax/2

rmax a property of the enzyme, interaction of E & S The rmax of an enzyme is a how fast the reaction it catalyzes can proceed once the enzyme-substrate complex is formed rmax =f ([E, T, pH ionic strength in solution]) rmax to the turnover number of substrate molecules converted into P per active site at very high substrate concentration.

Km, depends on the particular enzyme and substrate being used and on the conditions of temperature, pH, and ionic strength in the solution Km is independent of the enzyme concentration, in contrast to rmax. MM eq. plotted straight line to describe the kinetics of the enzyme under study.

Lineweaver-Burke plot
MM eq. converted to a linear form plotting 1/r (y) vs 1/[S] (x) called a double reciprocal plot or alineweaver-burke The equation of the line is:

the slope is KM/Vmax the intercept on the 1/r axis is 1/rmax

Example

From series of batch runs with a constant E concentration, the following initial rate data were obtained as a function of initial S concentration: Evaluate MM kinetic parameters using Lineweaver-Burk plot (do not use deviated data points from the MM model)

Data: S increase up to l0 mM the rate increased Further S increases (to 15mM) decreased the initial reaction rate may be due to substrate or product inhibition MM eq. does not incorporate the inhibition effects

S< 10mM rmax=0.54 mM/min & Km=1.78 mM All data rmax=0.45 mM/min & Km=1, 37 mM