Enzymes

Properties of Enzymes

General principles

1. The reaction catalysed by an enzyme uses exactly the same reactants and produces exactly the same products as the uncatalysed reaction. 2. Like other catalyst, enzymes do not alter the position of equilibrium between substrates and products. 3. However, unlike normal chemical reactions, enzymes are saturable. This means as more substrate is added, the reaction rate will increase, because more active sites become occupied. This can continue until all the enzyme becomes saturated with substrate and the rate reaches a maximum. 4. The two most important kinetic properties of an enzyme are how quickly the enzyme becomes saturated with a particular substrate, and the maximum rate it can achieve. Knowing these properties suggests what an enzyme might do in the environment of the cell and can show how the enzyme will respond to changes in these conditions.

Specificity of enzymes

Enzymes are usually very specific as to which reactions they catalyze and the substrates that are involved in these reactions. Complementary shape, charge and hydrophilic/hydrophobic characteristics of enzymes and substrates are responsible for this specificity. Enzymes can also show impressive levels of stereospecificity, regioselectivity and chemoselectivity. Some of the enzymes showing the highest specificity and accuracy are involved in the copying and expression of the genome. These enzymes have "proof-reading" mechanisms. Here, an enzyme such as DNA polymerase catalyzes a reaction in a first step and then checks that the product is correct in a second step. This two-step process results in average error rates of less than 1 error in 100 million reactions in highfidelity mammalian polymerases. Similar proofreading mechanisms are also found in RNA polymerase, aminoacyl tRNA synthetases and ribosomes. Some enzymes that produce secondary metabolites are described as promiscuous, as they can act on a relatively broad range of different substrates. It has been suggested that this broad substrate specificity is important for the evolution of new biosynthetic pathways.

How do enzymes work?

Like all catalysts, enzymes work by lowering the activation energy (Ea or G) for a reaction, thus dramatically increasing the rate of the reaction.

Free energy

The six major classes of enzimes

Oxidoreductases -catalyze oxidation-reduction reactions: 1. dehydrogenases 2. oxidases 2. reductases 3. peroxidase 4. oxygenase (monooxygenase, dioxygenases...)

Transferases -catalyze group-trasfer reactions Hydrolases -catalyze hydrolysis (esterases, phosphatases, peptidases) Lyases (Synthases) catalyze lysis of substrate, generating a double bond) Isomerases -catalyse structural change within a single molecule Ligases (Syntetases) -catalyze ligation or joining two substrates

"Lock and key" model

Enzymes are very specific, and it was suggested by Emil Fischer in 1894 that this was because both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. This is often referred to as "the lock and key" model. However, while this model explains enzyme specificity, it fails to explain the stabilization of the transition state that enzymes achieve. The "lock and key" model has proven inaccurate and the induced fit model is the most currently accepted enzyme-substratecoenzyme figure.

Induced fit model

In 1958 Daniel Koshland suggested a modification to the lock and key model: since enzymes are rather flexible structures, the active site is continually reshaped by interactions with the substrate as the substrate interacts with the enzyme. As a result, the substrate does not simply bind to a rigid active site, the amino acid side chains which make up the active site are moulded into the precise positions that enable the enzyme to perform its catalytic function. In some cases, such as glycosidases, the substrate molecule also changes shape slightly as it enters the active site. The active site continues to change until the substrate is completely bound, at which point the final shape and charge is determined.

Enzymes have active sites

Active site. Subsequent studies led to the concept of the active site, the region of an enzyme to which substrates and other essential molecules (cofactors or prosthetic groups) bind.
Some important features of the active site are: 1. It is very small relative to the overall size of the enzyme molecule.

2. At the active site, reactive groups that may be a considerable distance apart in the primary structure are brought together by protein folding.

3. Interactions among groups in the active site and the substrate usually involve the noncovalent interactions: hydrogen bonds, and electrostatic, van der Waals, and hydrophobic interactions. 4. These interactions often occur in a cleft or crevice in the folded polypeptide, from which water and other molecules are excluded.

A. Chemical kinetics

Relationships between product (P) formed in a unit of time (P/ t) Velocity (v) of the reaction
P

Rate of equation

= V = k[S]

k1

S
k-1

B. Enzyme kinetics
Enzyme binds supstrate in enzyme-substrate form. Single-substrate mechanism for an enzyme reaction. k1, k-1 and k2 are the rate constants for the individual steps.
For enzymatic reactions which exhibit simple Michaelis-Menten kinetics and in which product formation is the rate-limiting step (i.e., when k2 << k-1) KMk1/k1=Kd, where Kd is the dissociation constant (affinity for substrate) of the enzyme-substrate (ES) complex. However, often k2 >> k-1, or k2 and k-1 are comparable, in which case nothing can be said about the enzyme affinity from the Michaelis constant alone. The Michaelis constant can be defined as:

Progress curve for an enzyme-catalyzed reaction

[P] t
[P] [P]

Initial slope = v0 =

Progress curve at two different enzyme concentration in the presence of the high initial concentrations of substrate: [S] >> [E] In this case = the rate product formation depends on enzyme concentration and not on the substrate concentration.

The Michaelis Menten Equation

Michaelis-Menten kinetics describes the kinetics of many enzymes. It is named after Leonor Michaelis and Maud Menten.
This kinetic model is relevant to situations where the concentration of enzyme is much lower than the concentration of substrate (i.e. where enzyme concentration is the limiting factor), and when the enzyme is not allosteric.

Determination of constants

Saturation curve for an enzyme showing the relation between the concentration of substrate and rate.

To determine the maximum rate of an enzyme mediated reaction, the substrate concentration ([S]) is increased until a constant rate of product formation is achieved. This is the maximum velocity (Vmax) of the enzyme. In this state enzyme active sites are saturated with substrate. Note that at the maximum velocity, the other factors that affect the rate of reaction (ie. pH, temperature, etc) are at optimal values.

Reaction rate/velocity V

The speed V means the number of reactions per second that are catalyzed by an enzyme. With increasing concentration [S], the substrate enzyme is asymptotically approaching its maximum speed Vmax, but never actually reaching it. Because of that, no [S] for Vmax can be given. Instead, the characteristic value for the enzyme is defined by the substrate concentration at its half-maximum speed (Vmax/2). This KM value is also called the Michaelis-Menten constant.

Rate (or kinetic assay):

Measure concentration of analyte or product in initial stages only of the reaction (usually < 5 mins.) Determine INITIAL RATE (= SLOPE of the line as close as possible to start of reaction).

Measurement and Interpretation of Rate Automated instrument may make readings at two set times, say 1 min and 5 mins after initiating reaction.
Computes the rate between these two times C5 - C1
(but must be sure the concentration - time graph is close to linear over this time)

ie

5-1

TWO DISTINCT AND DIFFERENT PURPOSES OF RATE MEASUREMENT:

(i) ENZYME ASSAY in contrast to (ii) SUBSTRATE ASSAY using an enzyme reaction.

(i) ENZYME ASSAY Aim is to determine concentration of active enzyme, making use of the fact that rates of enzyme-catalysed reactions are proportional to the concentration of active enzyme. Require rate measurement not sensitive substrate concentration . So need [substrate] >> Km. Then kinetics are zero order with respect to substrate as rate approaches Vmax (Region 1 in Figure below). (ii) SUBSTRATE ASSAY Aim is to determine concentration of substrate by choosing conditions where rate is proportional to substrate concentration (ie 1st order). So need [substrate] << Km (Region 2 in Figure above).

Michaelis-Menten constant 'KM'

Since Vmax cannot be reached at any substrate concentration (because of its asymptotic behaviour, V keeps growing at any [S], albeit ever more slowly), enzymes are usually characterized by the substrate concentration at which the rate of reaction is half its maximum. This substrate concentration is called the Michaelis-Menten constant (KM). This represents (for enzyme reactions exhibiting simple Michaelis-Menten kinetics) the dissociation constant (affinity for substrate) of the enzyme-substrate (ES) complex. Low values indicate that the ES complex is held together very tightly and rarely dissociates without the substrate first reacting to form product. It is worth noting that KM can only be used to describe an enzyme's affinity for substrate when product formation is ratelimiting, i.e., when k2 << k-1 and KM becomes k-1/k1. Often, k2 >> k-1, or k2 and k-1 are comparable.

Derivation of the Michaelis-Menten Equation

This derivation of "Michaelis-Menten" was actually described by Briggs and Haldane. It is obtained as follows: The enzymatic reaction is supposed to be irreversible, and the product does not rebind the enzyme.

Because we follow the quasi steady state approximation. The concentrations of the intermediates are assumed to equillibrate much faster than those of the product and substrate, i.e. their time derivatives are zero:

Let's define the Michaelis constant:

This simplifies the form of the equation: (1)

The total (added) concentration of enzyme is a sum of that which is free in the solution and that which is bound to the substrate, and the free enzyme concentration is derived from this: [E0] = [E] + [ES] [E] = [E0] [ES] (2)
Using this concentration (2), the bound enzyme concentration (1) can now be written:

Rearranging gives:

(3)

The rate (or velocity) of the reaction is:

(4)

Substituting (3) in (4) and multiplying numerator and denominator by [S]:

This equation may be analyzed experimentally with a Lineweaver-Burk diagram or a Hanes-Woolf Plot.
E0 is the total or starting amount of enzyme. It is not practical to measure the amount of the enzyme substrate complex during the reaction, so the reaction must be written in terms of the total (starting) amount of enzyme, a known quantity. d[P]/dt a.k.a. V0 a.k.a. reaction velocity a.k.a. reaction rate is the rate of production of the product. Note that the term reaction velocity is misleading and reaction rate is preferred. k2[E0] a.k.a. Vmax is the maximum velocity or maximum rate. k2 is often called kcat.

This equation may be analyzed experimentally with a LineweaverBurk diagram or a Hanes-Woolf Plot.
The plot provides a useful graphical method for analysis of the Michaelis-Menten equation:

Taking the reciprocal gives:

V = reaction velocity (the reaction rate), Km = Michaelis-Menten constant, Vmax = maximum reaction velocity [S] is the substrate concentration.

Enzyme catalysis rates are affected by three important factors:

Temperature pH Concentration of substrate

Effect of temperature

This is not a true optimum, since the length of the assay will affect its value: A short assay will benefit from the increased rate, and not suffer too much from the exponential decay (over time) of enzyme activity at raised temperature. A longer assay will have more time for the enzyme to denature, so the observed 'optimum' will be lower.

The rate of any reaction increases with temperature. However, enzymes require a specific shape to work, and as the temperature increases, vibrations in the enzyme molecule will mess with its catalytic ability: at very high temperatures, the enzyme will completely denature (like egg white), becoming catalytically inactive. These competing processes lead to a temperature 'optimum' for the enzyme.

Effect of pH
Enzymes bristle with ionisable side groups with specific values of pKa (these may not be the values you would find for the isolated amino acid, as the environment of the side group will affect its pKa). If the active site contains a basic and an acidic amino acid that are required for catalysis, and both need to be ionised to interact with the substrate, then there will be a (true) optimum pH somewhere between the pKa of the acid and the pKb of the base. The relationship used to analyse this is usually: V = V0 ( 1 + ( 10pH Ka ) + ( Kb 10pH ) ) However, the dissociation constants Ka and Kb may be spurious under most circumstances, even though the equation usually fits the pH dependence curve nicely.

Effect of enzyme concentration on reaction rate

Effect of substrate concentration on reaction rate (hyperbolic)

An increase substrate concentration initially leads to a linear increase in reaction rate (first-order kinetics). This trend continues as long as the initial substrate concentration does not saturate or occupy all available active sites. As the concentration of substrate reaches levels where the active sites are saturated, the initial reaction rate starts to decrease (mixed-order kinetics). Eventually the substrate concentration is so high that it continuously keeps the active sites occupied and saturated, reaching a maximum initial velocity (zero-order kinetics at Vmax). Km on the graph indicates where half Vmax is reached. This type of kinetics is termed hyperbolic and is usually shown by simple, monomeric enzymes.

Units for expressing enzyme activity

Reaction rate implies substrate utilised per unit time or product formed per unit time. The katal is the SI unit but is not often used in ordinary conversation. It is defined as the transformation of mole of substrate per second.

Enzyme activity is defined as the amount of enzyme converting 1 m of

substrate per second.

Turnover number is another common term i.e. the number of substrate Specific activity refers to enzyme activity per mass of protein i.e. all

molecules converted by one enzyme molecule under specified conditions. the protein in a sample may not be enzyme. This unit also gives an indication of enzyme purity i.e. an impure enzyme will give low activity per unit mass.

Meaninig of Km

Michaelis constants have been determined for many of the commonly used enzymes. The size of Km tells us several things about a particular enzyme: 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. A Km of 10-7 M indicates that the substrate has a greater affinity for the enzyme than if the Km is 10-5 M.

1.

2.

Example of importance of Km is the physiological utilisation of glucose

Glucose can be phosphorylated by two different kinases to form glucose 6-phosphate. Liver contains both hexokinase and glucokinase that catalyse the identical reaction of:
glucose 6-phosphate + ADP

glucose + ATP

For hexokinase the Km for glucose is 0.1 mM, while for glucokinase it is 0.5 mM. When the concentration of blood sugar is low, as occurs in the fasted state, hexokinase is used to phosphorylate glucose; but when blood glucose increases after feeding, the high Km enzyme also functions.

Limitations

Michaelis-Menten kinetics, like other classical biochemical kinetic theories, relies on the law of mass action derived from the assumptions of free diffusion and thermodynamically-driven random collision. However, many biochemical or cellular processes deviate significantly from such conditions. For example, the cytoplasm inside a cell behaves more like a gel than a freely flowable/watery liquid, due to the very high concentration of protein (up to ~400 mg/mL) and other solutes, which can severely limit molecular movements (diffusion or collision). For heterogeneous enzymatic reactions, such as those of membrane enzymes, molecular mobility of the enzyme or substrates can also be severely restricted, due to the immobilization or phase-separation of the reactants. For some homogenous enzymatic reactions, the mobility of the enzyme or substrate may also be limited, such as the case of DNA polymerase where the enzyme moves along a chained substrate, rather than having a threedimensional freedom.

The Catalytic Constant kcat

At high substrate concentration the overall velocity of the reaction is Vmax and the rate is determined by the enzyme concentration. The rate constant observed under these conditions is called the catalytic constant, kcat, defined as:

kcat indicates

the maximum number of substrate molecules converted to product each second by each active site. This is called turnover number. The catalytic constant measures how fast a given enzyme can catalyze a specific reaction (describing the effectiveness of an enzyme) The unit for kcat is s-1 (for the most enzymes, kcat is 102 to 103 s-1)

Reversible Enzyme inhibition

Noncompetitive

Competitive

Uncompetitive

A. Competitive Inhibition

Vmax unchanged Km increases

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.

In competitive inhibition, both inhibitor and substrate can bind to enzyme and form two independent complexes. Only ES degrades to products: EI is considered a 'dead-end'. Because the inhibitor binds, to the active site, the substrate cannot (and vice versa), so there cannot be an ternary ESI complex.

Ki = [E] [I] [EI]

Ki is the dissociation constant for EI, like Km is the dissociation constant for ES.

Vmax unchanged Km increases

B. Uncompetitive Inhibition
Uncompetitive inhibition takes place when an enzyme inhibitor binds only to the complex formed between the enzyme and the substrate (the E-S complex), not the free enzyme. Mechanism This reduction in the effective concentration of the E-S complex increases the enzyme's apparent affinity for the substrate through Le Chatelier's principle (Km is lowered) and decreases the maximum enzyme activity (Vmax), as it takes longer for the substrate or product to leave the active site. Uncompetitive inhibition works best when substrate concentration is high.

Lowers Vmax and Km

C. Noncompetitive Inhibition

Lowers Vmax

Noncompetitive inhibitor can bind to E or ES, forming inactive EI or ESI complexes. These inhibitors are not substrate analogs and do not bind the same site as substrate.
Clasic noncompetitive inhibition is rare, but examples are known in allosteric enzymes.

Because the inhibitor can bind independently of the substrate, an ESI complex can also form. Both ESI and EI are dead-ends.

Ki = [E] [I] [EI] Kis = [ES] [I] [ESI]

Non-competitive inhibitors bind somewhere other than the active site, and these bindings reduce the speed at which the enzyme runs, reducing the apparent Vm. Km is unaffected, because for a non-competitive inhibitor, Ki = Kis, and if you run the maths, this results in no apparent change to Km.

Uses of Enzyme Inhibition

Enzyme inhibitors are found in nature and are also designed and produced as part of pharmacology and biochemistry.
Natural poisons are often enzyme inhibitors that have evolved to defend a plant or animal against predators. These natural toxins include some of the most poisonous compounds known. Artificial inhibitors are often used as drugs, but can also be insecticides (malathion), herbicides such as (glyphosate), or disinfectants (triclosan).

Two examples of a medicinal enzyme inhibitors

is sildenafil (Viagra), a common treatment for male erectile dysfunction. This compound is a potent inhibitor of cGMP specific phosphodiesterase type 5, the enzyme that degrades the signalling molecule cGMP (cyclic guanosine monophosphat) .
This signalling molecule triggers smooth muscle relaxation and allows blood flow into the corpus cavernosum, which causes an erection. Since the drug decreases the activity of the enzyme that halts the signal, it makes this signal last for a longer period of time.

The coenzyme folic acid (left) compared to the anti-cancer drug methotrexate (right)

Another example of the structural similarity of some inhibitors to the substrates of the enzymes they target is seen in the figure comparing the drug methotrexate to folic acid. Folic acid is the oxidised form of the substrate of dihydrofolate reductase, an enzyme that is potently inhibited by methotrexate. Methotrexate blocks the action of dihydrofolate reductase and thereby halts thymidine biosynthesis. This block of nucleotide biosynthesis is selectively toxic to rapidly growing cells, therefore methotrexate is often used in cancer chemotherapy.

Irreversible Enzyme Inhibition

Irreversible inhibitors usually covalently modify an enzyme, and inhibition cannot therefore be reversed. Irreversible inhibitors often contain reactive functional groups such as: 1. nitrogen mustards, 2. aldehydes (O=CH-) 3. haloalkanes 4. alkenes

These electrophilic groups react with amino acid side chains to form covalent adducts. The residues modified are those with side chains containing nucleophiles such as hydroxyl or sulfhydryl groups; these include the amino acids Ser, Cys, Thr and Tyr.

Example of irreversible inhibition

chymotrypsin
Diisopropyl fluorophosphate (DPF) (Organophosphorous nerve gas, inhibitor of hydrolases with the reactive serine as part of active site.)

(DFP)

chymotrypsin

The serine protease chymotrypsin is important digestive enzyme. Inhibition of acetylcholinesterase (catalyzes hydrolysis of of the neurotrasmitter acetylcholine) causes a paralysis.

Diisopropylphosphoryl-chymotripsin

Allosteric Enzymes

The enzyme has two binding sites, one for the substrate (the active site) and the other for the allosteric activator (the regulatory site). When the allosteric activator is not bound to the regulatory site, the active site of the enzyme is not able to bind substrate and catalyze the production of product. However, when the allosteric activator binds to the enzyme at the regulatory site, the shape of the active site changes so that it can bind its substrate and catalyze the production of products A and B. The enzyme will remain activated until the allosteric activator leaves the regulatory site.

Allosteric regulation is an important mechanism of hemoglobin

and is also used extensively in the control of enzyme activity

Allosteric means 'other site' and refers the the effect of small molecules that bind at sites other than the active site of the molecule. In hemoglobin, the molecule 2,3-bisphosphoglycerate (2,3BPG) is a negatively-charged allosteric effector that binds to a positively-charged central region, stabilizing hemoglobin in the T state and promoting unloading of oxygen in tissues.

General Properties of Allosteric Enzymes

Many enzymes do not demonstrate hyperbolic saturation kinetics, or typical Michaelis-Menten kinetics.

Graphs of initial velocity vs. substrate demonstrate sigmoidal dependency of v on S, much as we discussed with hemoglobin binding of dioxygen.

Enzymes that display this non Michaelis-Menten behavior have common characteristics. They :

are multi-subunit bind other ligands at sites other than the active site (allosteric sites) can be either activated or inhibited by allosteric ligands exist in two major conformational states, R and T often control key reactions in major pathways, which must be regulated

In allosteric activation, the activator locks the enzyme in the relaxed conformation

A typical allosteric inhibitor therefore cooperates in a heterotropic, negative fashion.

There is some vocabulary you should know for cooperativity: Positive: one molecule facilitates the binding of another. Negative: one molecule makes the binding of another more difficult. Homotropic: one molecule influences the binding of a second similar molecule. Heterotropic: one molecule influences the binding of a different molecule.

Multienzyme Complexes and Multifunctional Enzymes

In a number of metabolic pathways, several enzymes which catalyze different stages of the process have been found to be associated noncovalently, giving a multienzyme complex.
Examples: Pyruvate Dehydrogenase Complex; Electron Respiratory Chain

In other cases, different activities may be found on a single multifunctional polypeptide chain. The presence of multiple activities is on a single polypeptide chain is usually the result of a gene fusion event.

Mechanisms of Action

The catalytic power of enzymes depend on their ability to lower the activation barrier separating reactants and products. That is the energy required to cause a reaction to occur is lower when an enzyme is present. In order to achieve this enzymes:

May provide an environment favourable to the reaction, May cause intermediate reactions that step toward the final product, May act as acid/base catalysts (i.e. proton donors or proton acceptors), May covalently bond with the substrate and thus weaken other bonds within the substrate.

Properties of Serine Proteases

1. 2. 3. 4.

In biochemistry, serine proteases or serine endopeptidases are a class of peptidases (enzymes that cleave peptide bonds in proteins) that are characterised by the presence of a serine residue in the active site of the enzyme. Serine proteases are grouped into clans that share structural homology and then further subgrouped into families that share close sequence homology. The major clans found in humans include: chymotripsin-like, subtilisin-like, alpha/beta hydrolase, signal peptidase clans. Serine proteases participate in a wide range of functions in the body, including blood clotting, immunity, and inflammation, as well as contributing to digestive enzymes in both prokaryotes and eukaryotes.

Digestive serine proteases

Members: Chymotrypsin-clan

The three serine proteases of the chymotrypsin-like clan that have been studied in greatest detail are: Chymotrypsin Trypsin Elastase All three enzymes are synthesized by the pancreatic acinar cells, secreted in the small intestine and are responsible for catalyzing the hydrolysis of peptide bonds. All three of these enzymes are similar in structure, as shown through their X-ray structures. The differing aspect lies in the peptide bond which is being cleaved, which is called the scissile bond.

Each of these digestive serine proteases targets different regions of a polypeptide chain, based upon the side chains of the amino acid residues surrounding the site of cleavage: Chymotripsin is responsible for cleaving peptide bonds following a bulky hydrophobic amino acid residue. Preferred residues include Phe, Trp and Tyr, which fit into a snug hydrophobic pocket. Trypsin is responsible for cleaving peptide bonds following a positively-charged amino acid residue. Instead of having the hydrophobic pocket of the chymotrypsin, there exists an aspartic acid residue at the base of the pocket. This can then interact with positively-charged residues such as arginine and lysine on the substrate peptide to be cleaved.

Elastase is responsible for cleaving peptide bonds following a small neutral amino acid residue, such as Ala, Gly and Val. (These amino acid residues form much of the connective tissues in meat). The pocket that is in "trypsin" and "chymotrypsin" is now partially filled with valine and threonine, rendering it a mere depression, which can accommodate these smaller amino acid residues. The combination of these three enzymes make an incredibly effective digestive team, and are primarily responsible for the digestion of proteins.
Subtilisin Subtilisin is a serine protease in prokaryotes. Subtilisin is evolutionary unrelated to the chymotrypsin-clan, but shares the same catalytic mechanism utilising a catalytic triad, to create a nucleophilic serine. This is the classic example used to illustrate convergent evolution, since the same mechanism evolved twice independently during evolution.

Zymogens are the usually inactive precursors of an enzyme

If the digestive enzymes were active when synthesized, they would immediately start chewing up the synthesizing organs and tissues. Acute pancreatitis is such a condition, in which there is premature activation of the digestive enzymes in the pancreas, resulting in selfdigestion (autolysis). It also complicates postmortem investigations, as the pancreas often digests itself before it can be assessed visually. Zymogens are large, inactive structures, which have the ability to break apart or change into the smaller activated enzymes. The difference between zymogens and the activated enzymes lies in the fact that the active site for catalysis of the zymogens is distorted. As a result, the substrate polypeptide cannot bind effectively, and proteolysis does not occur. Only after activation, during which the conformation and structure of the zymogen change and the active site is opened, can proteolysis occur. There are certain inhibitors which resemble the tetrahedral intermediate, and thus fill up the active site, preventing the enzyme from working properly. Trypsin, a powerful digestive enzyme, is generated in the pancreas. Inhibitors prevent self-digestion of the pancreas itself.

Subtilisin
Subtilisin is a serine protease in prokaryotes.
Subtilisin is evolutionary unrelated to the chymotrypsin-clan, but shares the same catalytic mechanism utilising a catalytic triade, to create a nucleophilic serine. This is the classic example used to illustrate convergent evolution, since the same mechanism evolved twice independently during evolution.

Serine proteases use both the chemical and the binding modes of catalyzes

The active site of serine proteases contain a hydrogen-bonded Ser-Asp-His catalytic triade.
The Ser residue serves as covalent catalyst and the His residue serves as an acid-base catalyst. Anionic tetrahedral intermediates are stabilized by hydrogen bonds with the enzyme.

Why does the activity of enzymes vary with pH?

Changing the pH alters the ionization state of amino acid side chains that ionize: Lys, Arg, His, Asp and Glu So, if ionic bonds are important to structural stability then the shape of the enzyme will change and the functionality of the enzyme will change. This is a general phenomena - related to the overall 3-D structure of all enzymes.

pH optimum of an enzyme shown as the classic 'bell-shaped' curve