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BACKGROUND THEORY
MICROBIAL
INHIBITION
ENZYME INHIBITION
CELL FUNCTION
INHIBITION
COMPETITIVE INHIBITION
NON-COMPETITIVE INHIBITION


Understanding Spectrophotometry
Absorbance, A
The amount of light absorbed by a solution.
       A = log(Io/I)         Where Io = incident light    and    I = transmitted light


Optical Density, OD
The amount of light passing through a sample to a detector relative to the total amount of light available. Optical density includes absorbance of the sample plus light scatter from turbidity

Microbial Growth Inhibition

Microbial Inhibition involves the slowing of substrate utilization and growth rates (Equations 1 & 2, respectively below) by inhibitory compounds such as heavy metals, pesticides, and chlorinated solvents. Inhibitors generally affect microorganisms in one of two ways: A. Enzyme Inhibition or B. Cell Function Inhibition. In the case of Enzyme Inhibition, substrate utilization by a single enzyme is inhibited directly resulting in a loss of biomass. During Cell Function Inhibition, an important and necessary cell function such as respiration is negatively affected thereby reducing the utilization of a particular substrate, which in turn limits biomass production.

EQ. 1    Substrate Utilization

  • rut = Rate of substrate utilization (MsL-3T-1)
  • qm = Maximum specific rate of substrate utilization (MsMx-1T-1)
  • S = Concentration of rate limiting substrate (MsL-3)
  • Xa= Concentration of active biomass (MxL-3)
  • K = Substrate concentration giving (1/2) the maximum rate of substrate utilization(MsL-3)



EQ. 2    Microbial Growth

  • = Specific growth rate (T-1)
  • rut = Rate of substrate utilization (MsL-3T-1)
  • Y = True yield for cell synthesis (MsMx-1)
  • b = Endogenous decay coefficient (T-1)


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A. Enzyme Inhibition

The term enzyme inhibition encompasses those processes by which the normal activity of an enzyme is reduced or completely eliminated. There are several classes of inhibitor compounds but for the purpose of this course, only Reversible Inhibitors will be investigated. The key characteristic of reversible inhibition is that enzyme activity can be regenerated at low inhibitor concentrations.

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REVERSIBLE INHIBITION
There are two main categories of reversible enzyme inhibition: Competitive Inhibition , and Non-Competitive Inhibition .


i. Competitive Inhibition

In the case of competitive inhibition, the substrate [S] is prevented from binding and being degraded by its corresponding enzyme [E], by inhibitory compounds who compete with it to occupy the enzyme's active binding site .
Competitive Inhibition
FIGURE 1. Competitive Inhibition


The effect of competitive inhibition on Michaelis Menten enzyme kinetics is described by Equation 3.


EQ. 3
Rate Expression
Rate Expression for Competitive Inhibition
  • v = Specific growth rate (T-1)
  • vmax = Maximum specific growth rate (T-1)
  • I = Concentration of competitive inhibitor (MiL-3)
  • Ki = Competitive inhibition rate constant (MiL-3)
  • Km = Substrate concentration giving (1/2) the maximum
    enzyme reaction rate (MsL-3)



According to the above equation, the impact of this mode of inhibition on enzyme kinetics is primarily determined by the inhibitor and substrate concentrations. At high inhibitor concentrations [I] or relatively low substrate concentrations [S], the inhibitor can drastically alter the enzyme kinetics by occupying a significant number of active site locations, so much so that the observed rate of reaction, Vo, is reduced below the uninhibited reaction profile. This causes the value of the apparent Km to increase to Keff :

EQ. 4
Competitive Inhibition K Parameter
  • Keff = Effective Inhibition K Parameter
    = Substrate concentration giving (1/2) the maximum reaction rate (MsL-3)
  • Km = Substrate concentration giving (1/2) the maximum
    enzyme reaction rate (MsL-3)
  • I = Concentration of competitive inhibitor(MiL-3)
  • Ki = Competitive inhibition rate constant (MiL-3)



However, at high substrate concentrations [S] or relatively low inhibitor concentrations [I], the inhibtor will cause little disruption to normal enzyme function and the maximum inhibited velocity Vmax(i) still will still aproach the maximum uninhibited velocity Vmax.

Competitive Inhibition

FIGURE 2.  Competitive Inhibition Enzyme Kinetics


The increase in the apparent Km value to Keff has direct impact on the microbial growth and substrate-utilization kinetics. Similar to the Michaelis Menten equation, the larger K value in the denominator of the substrate utilization and in turn the microbial growth equation, initially impacts degradation/production at low substrate concentrations, but is uneffective at high substrate concentrations. At large [S] values, the rate reduction caused by a competitive inhibitor is completely offset due to the fact that qeff  remains equal to qmax.

Effective Rate of Substrate Utilization Keff = K(1 + I/Ki)

qeff = qm
Effective Microbial Growth Rate
Effective Rate of Substrate UtilizationEffective Microbial Growth Rate



EXAMPLE CASE.   As indicated by its name, the enzyme methane monooxygenase oxidizes methane to methanol. However, in the presence of the inhibitory compound trichloroethene, which is also capable of binding to the active site of the moonooxygenase enzyme, methane may be excluded from binding thereby reducing its rate of oxidation.
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ii. Non-Competitive Inhibition

Unlike competitive inhibition, non-competitive inhibition involves compounds that in no way resemble the enzyme's substrate compound. These compounds therefore do not bind to the enzyme at the active site but inhibit activity by binding to some other site and inducing the enzyme to undergo conformational changes. This in-activates the enzyme inabling it to bind to its natural substrate and participate in the ES complex.
Non-Competitive Inhibition
FIGURE 3. Non-Competitive Inhibition


The effect of competitive inhibition on Michaelis Menten enzyme kinetics is described by Equation 5.


EQ. 5
Rate Expression
Rate Expression for Non-Competitive Inhibition
  • v = Specific growth rate (T-1)
  • vmax = Maximum specific growth rate (T-1)
  • I = Concentration of competitive inhibitor(MiL-3)
  • Ki = Competitive inhibition rate constant (MiL-3)
  • Km = Substrate concentration giving (1/2) the maximum
    enzyme reaction rate (MsL-3)


Due to the fact that non-competitve inhibitors render their bound enzymes unrecoverable, even at high substrate [S] concentrations, there presence reduces the maximum possible complex concentration [ES]. As a result, as the concentration of inhibitor increases the value of Vmax decreases (See Figure 4. below). This phenomenon is taken into account by the (1 + I/Ki) term in the denominator of Equation 5. Since the remaining active enzyme molecules are unaltered, Km is unchanged.


Non-Competitive Inhibition

`FIGURE 4.  Non-Competitive Inhibition Enzyme Kinetics


The decrease in the qm value to qeff has direct impact on the microbial growth and substrate-utilization kinetics. Similar to the Michaelis Menten equation, the smaller qeff value in the numerator of the substrate utilization and in turn the microbial growth equation, also reduces degradation/production at all substrate concentrations.

Effective Rate of Substrate Utilization Keff = K

qeff <qm
Effective Microbial Growth Rate
Effective Rate of Substrate UtilizationEffective Microbial Growth Rate



EXAMPLE CASE.   The sulfhydryl groups (-SH) of cysteine a common amino acid found in enzymes, will bind with inhibitory metals such as Copper, Cu(II), Mercury, Hg(II), and Silver, Ag(I) which reduce their level of activity.

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B. Cell Function Inhibition

DECOUPLING INHIBITION
Some chemical compounds are capable of inhibiting microbial growth by disrupting an important cell function, thereby reducing biomass levels and in turn slowing substrate utilization. This type of inhibition is referred to as "decoupling". The ability of decoupling inhibitors to increase the permeability of the cytoplasmic membrane to protons, reduces the proton-motive force across the membrane, which drives the synthesis of ATP, the cell's primary energy carrier. This method of inhibition is well modelled by a decrease in the yield for cell synthesis from Y, to Yeff  and/or an increase in the endogenous decay parameter b, to beff  as described by the equations below:



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