II. SOIL ENZYMES AND ENZYME KINETICS
Required Readings:
Al-Turki, A.I. and W.A. Dick. 2003. Myrosinase activity in soil. Soil Sci. Soc. Am. J. 67:139145.
Frankenberger, W.T. and W.A. Dick. 1983. Relationships between enzyme activities and
microbial growth and activity indices in soil. Soil Sci. Soc. Am. J. 47:945-951.
Suggested Readings:
Tabatabai, M. A. 1982. Soil enzymes. p. 903-915, 943-947. In A. L. Page, R. H. Miller, and D.
R. Keeney (eds.), Methods of Soil Analysis, Part 2. American Society of Agronomy, Madison,
Wisconsin.
Dick, W. A. and M. A. Tabatabai. 1992. Significance and potential uses of soil enzymes. p. 95127. In F. Blaine Metting (ed.), Soil Microbial Ecology. Marcel Dekker, New York.
Ruggiero, P., J. Dec and J-M Bollag. 1996. Soil as a catalytic system. p. 79-122. In G. Stotzky
and J-M Bollag (ed.), Soil Biochemistry-Volume 9, Marcell Dekker, New York.
Proteins called enzymes catalyze the transformations of many chemical compounds in soil. Like
all catalysts, enzymes increase the rate of a chemical reaction without themselves undergoing
permanent alteration. Enzymes have unique properties, however, that distinguish them from
other catalysts. First, they are the most efficient catalysts known and are able to increase the
speed of a reaction a million fold or more compared to the same reaction carried out in the
absence of an enzyme. Second, enzymes are specific in the types of reactions in which they
participate. Third, enzymes are subject to regulation and thus act to control metabolite
concentrations and flow. Fourth, enzymes act under normal physiological conditions of
temperature and pressure.
The development of animal and plant biochemistry has been closely linked to the study of
enzymes with early enzyme-related research dealing primarily with fermentation reactions. Louis
Pasteur postulated in 1860 that fermentation was catalyzed by enzymes intimately associated
with the structure and life processes of the yeast cell. Edward Buchner demonstrated in 1897 that
enzymes involved in alcoholic fermentation could be extracted from yeast cells and could
operate independently of the cell structure. It was not until 1923, however, that Summer
accomplished the first isolation of an enzyme (urease).
The early history of soil enzyme research roughly parallels that of the animal and plant
biochemists. Woods in 1899 proposed that enzymes originating from, but existing outside, living
tissue might catalyze many organic matter transformations in soil. During the first decade of the
twentieth century several enzymatic activities including catalase, "oxydases", and a proteinase
were demonstrated in soil. Since that time the number of enzymes detected and studied in soil
has rapidly increased and is probably now well over 100.
Enzyme Nomenclature. The nomenclature of enzymes traditionally was arrived at by applying
the suffix ase to the name of the compound upon which the enzyme exerted its catalytic effect.
For example, urease catalyzed the conversion of urea to carbon dioxide and water. Other
enzymes were named after the type of chemical bond involved in the reaction or the type of
chemical change that occurred in a molecule as a result of the enzyme. Still other enzymes, such
as trypsin and pepsin, were given names that were nondescript and provided no clue to the type
of reaction that was catalyzed. To bring order to the confusion that resulted from all the names
formulated for the hundreds of enzymes discovered, the International Enzyme Commission
adopted a classification scheme in 1964 that is been continuously revised and updated
(http://www.chem.qmul.ac.uk/iubmb/enzyme/index.html). All enzymes are assigned to six main
classes based on the type of chemical reaction they participated in (Table 2.1). The main classes
are then broken down into subclasses and sub-subclasses. A biochemist working in any
discipline will often encounter the commonly used, or traditional names, for various enzymes. In
soil biochemistry literature, enzymes are often grouped as amidases, decarboxylases,
dehydrogenases, lipases, phosphatases, etc. This nomenclature has become so widespread that a
student of biochemistry must often be familiar with two names for the same enzyme.
Michaelis-Menten Equation. Modern kinetic theory proposes that for a chemical reaction to
occur, the reactants must first come together with sufficient energy to form an activated
transition state. Once sufficient energy has been attained to form the activated transition state, the
probability that the chemical reaction will proceed to the formation of product is much increased.
Enzymes are efficient catalysts because they bring about the formation of an activated transition
complex at a very low energy expense. An additional feature of a catalyzed reaction, however, is
the phenomenon of saturation. As the concentration of the substrate increases from zero, the
initial velocity of the reaction is observed to follow first-order kinetics. However, the rate of
increase in initial velocity becomes less and less with each unit of increase in substrate
concentration. Eventually a point is reached where no further increase in initial velocity occurs
and the reaction can be described by zero-order kinetics. Early investigators hypothesized that in
the initial step of an enzyme catalyzed reaction, the enzyme and substrate react reversibly to
form an enzyme-substrate complex. This hypothesis was very important in the formulation of the
general theory of enzyme kinetics proposed by Michaelis and Menten in 1913.
Table 2.1. International Enzyme Commission classification of enzymes (classes and partial list
of subclasses.
1.
Oxidoreductases (oxidation-reduction reactions)
1.1
1.2
1.3
1.4
1.5
1.6
Acting on > CH-OH functional groups
Acting on > C=O functional groups
Acting on > C=CH- functional groups
Acting on > CH-NH2 functional groups
Acting on > CH-NH- functional groups
Acting on NADH, NADPH
2. Transferases (reactions involving the transfer of a group of atoms from a donor to an
acceptor molecule)
2.1
2.2
2.3
2.4
2.7
2.8
One-carbon transfers
Aldehyde or ketone groups transfers
Acyl group transfers
Glycosyl group transfers
Phosphate group transfers
S-containing group transfers
3. Hydrolases (reactions involving the hydrolytic cleavage of bonds)
3.1
3.2
3.4
3.5
3.6
Hydrolysis of ester bonds
Hydrolysis of glycosidic bonds
Hydrolysis of peptide bonds
Hydrolysis of C-N bonds
Hydrolysis of acid anhydrides
4. Lyases (reactions involving the cleavage of bonds other than by hydrolysis or
oxidation)
4.1
4.2
4.3
4.4
Carbon-carbon bonds
Carbon-oxygen bonds
Carbon-nitrogen bonds
Carbon-sulfur bonds
5. Isomerases (reactions involving racemization, epimerization, and cis-trans
isomerization)
5.1 Racemization and epimerization
5.2 Cis-trans isomerization
6. Ligases (reactions involving the formulation of bonds by the cleavage of ATP)
6.1 Formation of C-O bonds
6.2 Formation of C-S bonds
6.3 Formation of C-N bonds
6.4 Formation of C-C bonds
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In developing the equation that bears their name, Michaelis and Menten made several
assumptions. The first two have already been mentioned and can be given as follows:
1. The rate of an enzyme catalyzed reaction changes from first-order to zero-order
kinetics.
2. Enzyme (E) reversibly binds with substrate (S) to form an intermediate
enzyme-substrate complex (ES) which then breaks down to form product (P).
Each reaction is described by a specific rate constant designated as K1, K2, and K3
k1
k3
E + S <---------- > ES ----------> E + P
k2
Four other assumptions were also very important in formulating the enzyme kinetic equation.
3. A steady state equilibrium between the rate of formation and the rate of
degradation of ES is rapidly achieved.
4. The concentration of total enzyme is defined as the concentration of enzyme
in the free state and the concentration of the enzyme-substrate complex.
5. The rate-limiting step is the decomposition of the enzyme-substrate complex to
form product. The initial velocity of the enzyme-catalyzed reaction is thus
proportional to the concentration of the enzyme-substrate complex
6. The maximum rate of reaction (Vmax) is attained when the concentration of the
7. enzyme-substrate complex reaches a maximum. This will occur only when all
the free enzyme becomes complexed with substrate, i. e. saturation.
Given these assumptions, the classical form of the Michaelis-Menten equation can be obtained.
Vi = Vmax(S)/km + (S)
Km is called the Michaelis constant that is really a combination of the individual reaction
constants (k1, k2, and k3) of the enzyme-catalyzed reaction. Knowledge of the Km value
provides useful information regarding the interaction of the enzyme with its substrate. The
smaller the Km value, the greater is the affinity of the enzyme for its substrate. The value is
easily determined using graphical techniques that plot the inverse of the initial velocity of an
enzyme-catalyzed reaction on the y-axis against the inverse of the substrate concentration.
Soil Enzyme Activity. All enzyme activity in soil is ultimately derived from microorganisms,
plants, or soil animals with a distinction often being made between activity originating from
extracellular enzymes and enzymes associated with metabolizing and proliferating microbial
cells (intracellular enzymes). Both constitutive and inducible enzymes are present in the soil.
Constitutive enzymes are those that are present in nearly constant amounts in a cell and their
activity is not affected by the addition of any particular substrate. Inorganic pyrophosphatase is
an example of a constitutive enzyme. Inducible enzymes are those present only in trace amounts
in a cell but which can quickly increase in concentration when its substrate is present. Inducible
enzymes play an important role in regulating the concentration of various commonly
encountered compounds added to soil. Many polysaccharidases, such as cellulase, are inducible
enzymes.
Basic information is still lacking, however, concerning:
1) The source and nature of enzyme activity in soil. This problem involves
development of modern technologies for the assay of enzyme activity in soil
and the extraction and characterization of soil enzymes.
2) The importance of the behavior of enzyme systems in soil ecosystems.
This area of research involves study of the transfer of genetic material which codes for a specific
enzyme and the soil factors that affect the expression of that enzyme, the role of soil enzymes in
the metabolism of xenobiotics and metal pollutants, and enzyme activity in the plant root
rhizosphere.
Soil is by no means an inert material. A vast array of microorganisms, plants, and animals
manufacture, degrade, and/or transform a countless number of different chemical compounds.
These reactions are mediated by enzymes either within the living cell or by extracellular
enzymes. The study of soil enzymes can yield information that is vital to our understanding of
the total soil system.