Soil Enzymes

Reading Assignment Soil Microbiology: An exploratory Approach

Chapter 3

Soil Enzymes: 1. Introduction

Nutrient cycling in soils involves biochemical, chemical, and physicochemical reactions with the biochemical processes being mediate by soil microbes.

Soil Enzymes: 1. Introduction

Biochemical reactions are catalyzed by enzymes, which are proteins with catalytic properties owing to their power of specific activities. Enzymes are catalysts, that is, they are substances which without undergoing permanent alteration cause chemical reactions to proceed at faster rates.

Soil Enzymes: 1. Introduction

First enzyme isolated was urease in 1923 by Sumner Now about 2,000 have been isolated and 25 have 3 dimensional structure determined.

Enzymes: Properties
Enzymes Distinguished from other Catalyst by: 1. Extremely efficient Under optimum conditions, they can catalyze reactions at rates that are 108 to 1019 times (up to 10 billion times) more rapid than those of comparable reactions

Enzymes: Properties
without enzymes and up to106 fold more the normal chemical catalyst. The turnover number (number of substrate molecules metabolized per enzyme molecule per second) is generally between 1 and 10,000 and in some instances may be as high as 500,000

Enzymes: Properties

2. Specificity of Reaction. For example, a specific enzyme may be capable of hydrolyzing a peptide bond only between two specific amino acids. Enzyme specificity results from the threedimensional shape of the active site, which fits substrate somewhat like a lock with its key.

Enzymes: Properties

Enzyme specificity is dictated by the nature of the groups attached to the susceptible bonds, e.g. Maltase hydrolyzes maltose to glucose, whereas cellobiase hydrolyzes cellubiose to glucose but not vice versa. Differences between the 2 substrates is slight in that maltase is an -glucoside and cellulose is a -glucoside.

Enzymes: Properties
3. Subject to Cellular Regulation Thus act to control metabolite concentration and flow.

II. Enzyme Classification

A. International Enzyme Commission (Classes): Divides enzymes into six major classes and sets of subclasses according to the type of reaction they catalyze. Each enzyme is assigned a recommended name, a systematic name and a classification number.

II. Enzyme Classification

1. Oxidoreductases :(oxidationreduction reactions

e.g. CH3CH2OH + NAD+ CH3CHO + NADH (H)+ 1.1 Acting on CH-OH functional groups 1.2 Acting on C=O functional groups 1.3 Acting on C=CH- functional groups 1.4 Acting on CH-NH2 functional groups 1.5 Acting on CH-NH- functional groups 1.6 Acting on NADH, NADPH

II. Enzyme Classification

2. Transferases: Reactions involving transfer of a functional from donor to an acceptor molecule. e.g. Creatine + ATP Phosphocreatine + ADP
2:1 2:2 2.3 2.4 2.5 2.6 One carbon group transfers Aldehyde or ketonic group transfers Acyl groups transfers Glycosyl group transfers Phosphate group transfers Sulfur group transfers

II. Enzyme Classification

3. Hydrolases: reactions involving the hydrolytic cleavage of bonds. e.g. Urea + H20 NH3 + CO2 3.1 Hydrolysis of Esters 3.2 Hydrolysis of Glycosidic bonds 3.4 Hydrolysis of Peptide bonds 3.5 Hydrolysis of C-N bonds 3.6 Hydrolysis of Acid Anhydrides

II. Enzyme Classification

4. Lyases (Addition to double bonds): Reactions involving the cleavage of bonds other than by hydrolysis or oxidation. e.g. Aspartic acid Alanine 4.1 C=C 4.2 C=O 4.3 C=N 4.4 C=S

II. Enzyme Classification

5. Isomerases: Reactions involving isomerization such as racemization, epimerization, and cis and trans isomerization. e.g. 5.1 Racemases 5.2 cis-trans isomerizations

II. Enzyme Classification

6. Ligases: Reactions involving the formation of bonds by the cleavage of ATP. 6.1 C-O bonds 6.2 C-S 6.3 C-N 6.4 C-C

II. Enzyme Classification

B. Based on Substrate Many enzymes are named by adding the suffix ase to the name of the substrate i.e. the molecule on which the enzyme exerts catalytic action, e.g. Cellulase - acts on cellulose Chitinase - acts on chitin Urease - catalyses hydrolysis of urea to ammonia and CO2.

II. Enzyme Classification

C. Based on the Substrate and Type of Reaction e.g. Succinic Dehydrogenase - removes hydrogen from succinic acid.

II. Enzyme Classification

D. Based on Site of Action. 1. Intracellular enzymes - perform functions within the confines of the cell. 2. Extra cellular enzymes - Concerned with reactions outside of the organism that synthesize the catalyst.

II. Enzyme Classification

E. Distinction based on when they are produced. 1. Constitutive enzymes - Always produced by the cell 2. Inducible enzyme - Formed only in the presence of specific substrate.

II. Enzyme Classification

F. Classified on basis of catalytic activity. 1. Those that depend on their protein structure only. 2. Those that require ions or one or more heat stable non protein molecules called cofactors.

II. Enzyme Classification

a. The cofactor may be a metal ion e.g. Fe, Cu, Mg2+, Co, Ca2+ and Zn2+ or an organic molecule called a coenzyme, some enzymes require both.
Many coenzymes are derived from vitamins. Two of the most important coenzymes are NAD and NADP Both compounds contain derivatives of the vitamin B nicotinic acid reactions i.e. removal of CO2.

II. Enzyme Classification

b. The catalytically active enzyme cofactor complex is called a haloenzyme. c. The Protein minus cofactor is called an apoenzyme.

II. Enzyme Classification

d. When the Coenzyme is tightly bonded to their apoenzyme, they are called prosthetic groups. e.g. heme (iron containing) group of an enzyme called cytochrome C
Cytochromes are a group of enzymes that function as electron carriers in respiration and photosynthesis.

II. Enzyme Classification

Cofactor function by: 1. Being the primary catalytic center 2. Acting as the bridging group to bind substrate and protein in a proper spacial alignment for catalytic activity to take place. 3. Complexing with the protein to stabilize the active three dimensional conformation state of the enzyme.

II. Enzyme Classification

4. Participating in the overall reaction by transferring a chemical group from the primary substrate to the second substrate. Metal ions operate by all four mechanisms. Organic coenzymes generally operate using the fourth mechanism

III. Mechanism of Enzyme Action

Although scientists do not fully understand how enzymes lower activation energy, the sequence of events is believed to be as follows:
1. The surface of the substrate- that is, the molecule or molecules that are the reactants in the chemical reaction to be catalyzed-contacts a specific region on the surface of the enzyme molecule, called the site.

III. Mechanism of Enzyme Action

2. A temporary intermediate compound called the enzyme-substrate complex forms. 3. The substrate molecule is transformed by rearrangement of existing atoms, a breakdown of existing molecule, or the combining of several substrate molecules.

III. Mechanism of Enzyme Action

4. The transformed substrate molecules, the products of the reaction, move away from the surface of the enzyme molecule. 5. The recovered enzyme molecule, now freed, reacts with other substrate molecules.

IV. Enzyme Kinetics

Enzymes catalyzed reactions exhibit kinetic properties similar to the chemical reactions. An additional feature of enzyme catalyzed reaction, whether enzymatic or nonenzymatic, is the phenomenon of saturation. As the concentration for the substrate increases from zero, the initial velocity (Vo) of the reaction is observed to be first-order.

IV. Enzyme Kinetics

The rate of increase in initial velocity becomes less and less with each unit increase in substrate concentration. Eventually a point is reached where no further increase in initial velocity occurs when substrate concentration increased and the reaction is can be described by zero-order kinetics This hypothesis was very important in formulating the general theory of enzyme kinetics, proposed by Michaelis and Menten in 1913.

Michaelis Menten Equation

Michaelis Menten Equation:

V = Vmax [S] Km + [S]

Where V Vmax [S] Km
= = = = the initial velocity the maximum rate of reaction the substrate concentration Micahelis-Menten constant

Michaelis Menten Equation

It relates initial velocity, maximal velocity and initial substrate concentration through the Michaelis-Menten constant. The Michaelis constant Km is equal to substrate concentration when the initial velocity is half the maximal rate.

Michaelis Menten Equation

1/Km is called the binding constant The larger the Km value, the less binding The smaller the Km value, the higher binding

Michaelis Menten Equation

Line Weaver Burke Transformation

1/V = Km /Vmax 1/[S] + 1/Vmax

When 1/V is plotted against 1/[S], you get a straight line It allows accurate determination of Vmax

Michaelis Menten Equation

Eadie-Hofstee Plot Vo = -Km Vo/[S] + Vmax A plot of Vo against Vo/[S], called the EadieHofstee plot. It not only yields Vmax in a very simple way but also magnifies departures from linearity which might not be apparent in a double reciprocal plot.

V. Factors Affecting Enzyme Action

1.

Substrate Concentrations There is a certain maximum rate at which a given amount of enzyme can catalyze a specific reaction. Only when the concentration of substrates is extremely high can this maximal rate be attained.

V. Factors Affecting Enzyme Action

Under conditions of high substrate concentrations the enzyme is said to be saturated, that is, its active site is occupied by substrate or product molecules at all times. When this happens, further increase in substrate concentration will have no effect on the reaction rate because additional active sites are not available for reaction.

V. Factors Affecting Enzyme Action

2. Enzyme Concentrations If substrate concentration exceeds saturation levels only additional enzyme production by a cell can further increase the rate of reaction. At any given time, many of the enzyme molecule are inactive for lack of substrate; Thus reaction rate is more likely to be determined by the substrate concentration

V. Factors Affecting Enzyme Action

The rate of most enzymatic reactions approximately doubles for each every 10oC rise in temperature (Q10 = 2) The temperature coefficient Q10, however, varies somewhat from one enzyme to another, depending on the energy activation and the catalyzed reaction.

V. Factors Affecting Enzyme Action

3. pH pH dependence of enzyme reactions is the consequence of changing degrees of ionization of groups in the enzyme, in the substrate, or in both. Most enzyme have characteristic pH at which their activity is maximal, this is referred to as the optimum pH.

V. Factors Affecting Enzyme Action

4. Temperature Most chemical reactions occur rapidly as the temperature rises. In enzymatic reactions, however, once a certain temperature is attained, any further elevation results in drastic decline in the reaction rate. This decrease in reaction is due to denaturation of the enzyme, a phenomenon common to all proteins.

V.

Factors Affecting Enzyme Action

5. Inhibitors Based on the mechanism of action, enzyme inhibitors are classified in two ways: competitive inhibitors and noncompetitive inhibitors. a. Competitive inhibitors compete with normal substrates for active sites.

Factors Affecting Enzyme Action

The competitive inhibitor is able to do this because its shape and chemical structure are similar to the normal substrate. An example of this is sulfanilamide (a sulfur drug). Para-benzoic acid is the normal substrate of the enzyme inhibited by sulfanilamide.

V. Factors Affecting Enzyme Action

It is the essential nutrient used by bacteria in the synthesis of folic acid, a vitamin that functions as a coenzyme. b. Non-competitive inhibitors do not compete with the substrate for the enzyme molecules activate site. Instead they act on other parts of the enzyme and indirectly decreases the ability of the normal substrate to combine with the enzyme.

V. Soil Enzymes

Importance of Soil Enzymes 1. Release of nutrients to the environment e.g. urease breaks down urea to NH3 which is a plant nutrient. 2. Identification of soils 3. Indication of microbial activity 4. Sensitive indicator of ecological changes.

B. Origin of Soil Enzyme Activity

1. Soil microorganisms - living and dead 2. Plant roots and residue. 3. Soil animals. Poor correlation between microbial numbers and enzyme activity . Microbial activity difficult to measure

B. Origin of Soil Enzyme Activity

Actively secreted by microbes and roots. Remain in decaying organic tissue. Made up of extra cellular and intracellular enzyme.

C. Location of Soil Enzyme Activity

1. Associated with bacteria cell walls, capsule of bacteria, cytoplasmic debris from lysed cells, the root-soil interphase, root cell walls, plant and microbial cell remnants and nonspecific materials found in soils.

C. Location of Soil Enzyme Activity

2. May be also associated with discrete soil particle size fractions Primarily associated with the clay fraction, while less activity with the silt and little activity with sand.

3. Enzyme proteins are readily adsorbed by clay minerals . The activity of adsorbed enzyme is affected by the type of clay mineral and the enzyme being studied, e.g. montmorrillonite clay has a large surface area and tends to adsorb more enzyme than kaolinite , a clay with less surface area.

D. State of Enzyme in Soil

Soil enzymes are very persistent i.e. very stable. Has been found to persist for 1000 yrs. e.g. Urease and Phosphatase have been demonstrated to be 9,000 yrs old in permafrost in Alaska. Phosphatase and Catalase between 4,000 and 10,000 yrs old have been observed in old lake sediments in Romania. Very resistant to destruction

E. Theories of Enzyme Stability in Soils.

Several theories have been suggested for the stability of soil enzymes in soils. Several of the mechanisms suggested involve the interaction of enzyme protein with clay minerals, organic matter and clay organic-matter complexes.
1. Role of Clays: a. Most activity associated with clay fraction of soil.

D. Theories of Enzyme Stability in Soils

b. Adsorption of proteins readily occurs. c. Are more resistant to proteolysis and microbial attack d. Form ESI-complexes which becomes stabilized but lower in activity . e. Increases inactivation temperature by 10oC

D. Theories of Enzyme Stability in Soils

2. Role of Organic Matter: Much of the information dealing with this hypothesis has been derived from studies with synthetic polymer enzyme complexes. Traps protein molecule in tertiary structure thus stabilizing the protein. a. Humus material known to stabilized to soil-N compounds e.g. protein.

D. Theories of Enzyme Stability in Soils

b. Enzyme attached to insoluble organic matrices exhibit pH and temperature changes, specificity change, like soil enzyme. c. Protected by globular humus complexes. d. Inability to purify soil enzymes free of soil organic matter.

D. Theories of Enzyme Stability in Soils

3. Role of Organic Matter-Clay Complexes: a. Lignin plus bentonite (clay) protect enzyme against proteolytic attack but not bentonite alone. b. Enzyme bound to organic matter which is bound to clay.

F. Measurement of Soil Enzyme Activity

1. Measured in similar fashion to that of other enzymes except for the problem of the existence of viable enzymes. 2. Problems In Methodology: a. Cannot separate extracellular from intracellular enzyme activity. b. Cannot measure specifically free, accumulated enzyme activity in soils.

F. Measurement of Soil Enzyme Activity

c. Cannot separate chemical from biological catalysis. Cannot overcome by setting appropriate control. d. Storage and treatment of sample greatly affect activity.

F. Measurement of Soil Enzyme Activity

Methods of Sterilization. a. Sterilization Kills microbes; may denature enzyme b. Irradiation: Kills live cells without disrupting enzymes; Effective Chemical and physical properties changes often negligible.

F. Measurement of Soil Enzyme Activity

Not used much today because: 1. Only few sources available; 2. Danger of radiation itself; 3. Difficulty in interrupting results. e.g. a. some enzyme not affected e.g. urease b. others greatly affected e.g. trypsin

F. Measurement of Soil Enzyme Activity

c. Chemical Agents (e.g. toluene, sodium azide). 1. Stops microbial synthesis of enzymes by living cells. 2. Prevents assimilation of product by enzymatic reaction. 3. May act as plasmolytic agent releasing contents i.e. intracellular enzyme (negative effect). 4. Acts as unmasking agent for activity of some enzymes

F. Measurement of Soil Enzyme Activity

4. Separation of Extra cellular from Intracellular Enzymes.

a. Sterilization - measures total enzyme activity b. Irradiation -measures soil enzyme activity c. Chemical Agents - measure extra-cellular enzyme

G. Application of Soil Enzyme Assays

1. Correlation with soil fertility. Difficult to show correlation especially fertility with enzyme activity. 2. Use to correlate microbial activity. Used as index of soil microbial population Enzyme which correlated best 3, amides, peroxidase and alkaline phosphatase.

G. Application of Soil Enzyme Assays

3. Correlate with biochemical cycling of various elements in soil (C, N , S ). has met with only limited success. 4. Degree of pollution Impact of numerous chemicals on the biological cycling of elements in soil can also be easily and sensitively assessed by determining their effect on soil enzyme activity. Nitrification is one of the sensitive assay toward pollution i.e. sensitive to acidity NH4 +, NO3-

G. Application of Soil Enzyme Assays

5. Forensic Purposes Can determine where soil comes from by looking at activity, Km value of sample size. 6. To Assess Ecological Succession As ecosystem changes the enzyme activity changes and this is due to O.M. changes. 7. Degradation of Herbicides? e.g. Enhance herbicide degradation.