Ph, temperature, enzyme concentration, substrate concentration, Derivation of

Michaelis - Menten equation for uni-substrate reactions

 Factors Affecting Enzyme Activity
Knowledge of basic enzyme kinetic theory is important in enzyme analysis in order both to
understand the basic enzymatic mechanism and to select a method for enzyme analysis.
The conditions selected to measure the activity of an enzyme would not be the same as
those selected to measure the concentration of its substrate. Several factors affect the rate
at which enzymatic reactions proceed - temperature, pH, enzyme concentration, substrate
concentration, and the presence of any inhibitors or activators.

 Enzyme Concentration
In order to study the effect of increasing the enzyme concentration upon the reaction rate,
the substrate must be present in an excess amount; i.e., the reaction must be independent
of the substrate concentration. Any change in the amount of product formed over a
specified period of time will be dependent upon the level of enzyme present. Graphically this
can be represented as:

These reactions are said to be "zero order" because the rates are independent of substrate
concentration, and are equal to some constant k. The formation of product proceeds at a
rate which is linear with time. The addition of more substrate does not serve to increase the
rate. In zero order kinetics, allowing the assay to run for double time results in double the
amount of product.

Table I: Reaction Orders with Respect to Substrate Concentration

Order Rate Equation Comments

Zero rate = k rate is independent of substrate concentration

First rate = k[S] rate is proportional to the first power of substrate concentration

Second rate = k[S][S]=k[S]2 rate is proportional to the square of the substrate concentration

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 Table I: Reaction Orders with Respect to Substrate Concentration

Order Rate Equation Comments

Second rate = k[S1][S2] rate is proportional to the first power of each of two reactants

The amount of enzyme present in a reaction is measured by the activity it catalyzes. The
relationship between activity and concentration is affected by many factors such as
temperature, pH, etc. An enzyme assay must be designed so that the observed activity is
proportional to the amount of enzyme present in order that the enzyme concentration is the
only limiting factor. It is satisfied only when the reaction is zero order.

In Figure 5, activity is directly proportional to concentration in the area AB, but not in BC.
Enzyme activity is generally greatest when substrate concentration is unlimiting.

When the concentration of the product of an enzymatic reaction is plotted against time, a
similar curve results, Figure 6.

Between A and B, the curve represents a zero order reaction; that is, one in which the rate
is constant with time. As substrate is used up, the enzyme's active sites are no longer
saturated, substrate concentration becomes rate limiting, and the reaction becomes first
order between B and C.

To measure enzyme activity ideally, the measurements must be made in that portion of the
curve where the reaction is zero order. A reaction is most likely to be zero order initially
since substrate concentration is then highest. To be certain that a reaction is zero order,
multiple measurements of product (or substrate) concentration must be made.

Figure 7 illustrates three types of reactions which might be encountered in enzyme assays
and shows the problems which might be enountered if only single measurements are made.

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B is a straight line representing a zero order reaction which permits accurate determination
of enzyme activity for part or all of the reaction time. A represents the type of reaction that
was shown in Figure 6. This reaction is zero order initially and then slows, presumably due
to substrate exhaustion or product inhibition. This type of reaction is sometimes referred to
as a "leading" reaction. True "potential" activity is represented by the dotted line. Curve C
represents a reaction with an initial "lag" phase. Again the dotted line represents the
potentially measurable activity. Multiple determinations of product concentration enable
each curve to be plotted and true activity determined. A single end point determination at E
would lead to the false conclusion that all three samples had identical enzyme
concentration.

 Substrate Concentration

It has been shown experimentally that if the amount of the enzyme is kept constant and the
substrate concentration is then gradually increased, the reaction velocity will increase until
it reaches a maximum. After this point, increases in substrate concentration will not
increase the velocity (∆A / ∆T). This is represented graphically in Figure 8.

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It is theorized that when this maximum velocity had been reached, all of the available
enzyme has been converted to ES, the enzyme substrate complex. This point on the graph
is designated Vmax. Using this maximum velocity and equation (7), Michaelis developed a
set of mathematical expressions to calculate enzyme activity in terms of reaction speed
from measurable laboratory data.

The Michaelis constant Km is defined as the substrate concentration at 1/2 the maximum
velocity. This is shown in Figure 8. Using this constant and the fact that Km can also be
defined as:

Km=K-1 + K2 / K+1

K+1, K-1 and K+2 being the rate constants from equation (7). Michaelis developed the
following

Michaelis constants have been determined for many of the commonly used enzymes. The
size of Km tells us several things about a particular enzyme.

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  A small Km indicates that the enzyme requires only a small amount of substrate to
become saturated. Hence, the maximum velocity is reached at relatively low
substrate concentrations.
 A large Km indicates the need for high substrate concentrations to achieve maximum
reaction velocity.
 The substrate with the lowest Km upon which the enzyme acts as a catalyst is
frequently assumed to be enzyme's natural substrate, though this is not true for all
enzymes.

 Temperature Effects

 Like most chemical reactions, the rate of an enzyme-catalyzed reaction increases as

the temperature is raised. A ten degree Centigrade rise in temperature will increase
the activity of most enzymes by 50 to 100%. Variations in reaction temperature as
small as 1 or 2 degrees may introduce changes of 10 to 20% in the results. In the
case of enzymatic reactions, this is complicated by the fact that many enzymes are
adversely affected by high temperatures. As shown in Figure 13, the reaction rate
increases with temperature to a maximum level, then abruptly declines with further
increase of temperature. Because most animal enzymes rapidly become denatured at
temperatures above 40°C, most enzyme determinations are carried out somewhat
below that temperature.
 Over a period of time, enzymes will be deactivated at even moderate temperatures.
Storage of enzymes at 5°C or below is generally the most suitable. Some enzymes
lose their activity when frozen.

 Effects of pH
 Enzymes are affected by changes in pH. The most favorable pH value - the point
where the enzyme is most active - is known as the optimum pH. This is graphically
illustrated in Figure 14.

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 Extremely high or low pH values generally result in complete loss of activity for most
enzymes. pH is also a factor in the stability of enzymes. As with activity, for each
enzyme there is also a region of pH optimal stability.
 The optimum pH value will vary greatly from one enzyme to another, as Table II
shows:

Table II: pH for Optimum Activity

Enzyme pH Optimum

Lipase (pancreas) 8.0

Lipase (stomach) 4.0 - 5.0

Lipase (castor oil) 4.7

Pepsin 1.5 - 1.6

Trypsin 7.8 - 8.7

Urease 7.0

Invertase 4.5

Maltase 6.1 - 6.8

Amylase (pancreas) 6.7 - 7.0

Amylase (malt) 4.6 - 5.2

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 Table II: pH for Optimum Activity

Enzyme pH Optimum

Catalase 7.0

 In addition to temperature and pH there are other factors, such as ionic strength,
which can affect the enzymatic reaction. Each of these physical and chemical
parameters must be considered and optimized in order for an enzymatic reaction to
be accurate and reproducible.

 DERIVATION OF THE MICHAELIS-MENTEN EQUATION FOR ENZYME KINETICS

Michaelis-Menten equation, which gives the velocity v of the reaction in terms of the substrate
concentration [S]:

  (1)

The use of this equation implies several assumptions, which require a bit of explanation. The reaction
scheme below is often referred to as the Michaelis-Menten reaction:

  (2)

In this scheme, an enzyme combines with a substrate in a reversible interaction to form an enzyme-
substrate complex (ES, in equation 2). This complex persists for a short time and then either dissociates
into enzyme and substrate, or the substrate is changed into a product P. The product in this scheme
is assumed to have no affinity for the enzyme, so it detaches. Thus the second part of the reaction is
irreversible formation of product accompanied by regeneration of the active enzyme.

To derive equation 1 from the reaction scheme 2 we start from an expression for the velocity v which
follows from scheme 2, which is:

  (3)

Here we assume that the overall reaction velocity is limited by the product formation step, so that the
overall rate is given by the rate of that step, i.e. the product of the concentration of enzyme-substrate
complex and the rate constant k2.

Now we obtain more useful expressions for both k2 and [ES], but we must make a couple more
assumptions to do so.

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We now assume that the reaction is in a state where the amount of complex [ES] is nearly constant, with
the rate of formation equal to the rates of dissociation - both backward and forward. These rates are
expressed as in equation 3 and we now assert that

  (4)

Some rearrangement gives us equation 5:

  (5)

We simplify equation 5 by combining the three rate constants into one, which is called Km, the Michaelis-
Menten rate constant, thus:

  (6)

This allows us to rewrite equation 5 this way:

  (7)

Now we want to eliminate [E] from this expression. To do this we use the fact that the total amount of
enzyme in the system doesn't change, since it is a catalyst and is always regenerated after each reaction.
(This 'fact' is actually another assumption, one which we will not discuss here.) We express that fact in
equation 8, where [E0] is the total (or equivalently initial) enzyme concentration:

  (8)

When equation 8 is solved for [E], we can eliminate [E] from equation 7 by substitution:

  (9)

Now rearrange and solve equation 9 for [ES] to obtain an expression which depends only on [S]:

  (10)

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When equation 10 is substituted into equation 3, we are almost finished:

  (11)

We now come to the last important assumption necessary to arrive at equation 1. Observe that the
maximum rate for reaction 2 occurs when the greatest possible proportion of the enzyme is combined
with substrate all the time, which occurs when [S] >> [E].  If we assume that this is the case, then we can
write

  (12)

just as we did to obtain equation 3. Substituting equation 12 into equation 11 produces the desired result
- equation 1, the Michaelis-Menten equation:

   

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