Effect of Physical and Chemical Agents on Kinetics | Enzymes

We will first consider the effects of the variations of two physical factors, temperature and pH. We will then examine certain chemical agents (or effec­tors) which can modify the kinetics of enzymatic reactions and are either activators or inhibitors. 

1. Effect of Temperature:

The study of the initial velocity of an enzymatic reaction as a function of temperature reveals two very distinct phases corresponding to two different phenomena (see fig. 2-6):

a. In the zone of the lowest temperatures (in figure 2-6, between 0 and 40° approximately) reaction velocity increases when temperature increases. This increase of velocity with temperature is simply explained as seen earlier, by an increase in activated complex concentration, when more energy in thermal form is supplied to the reacting system.

b. Then, beyond a certain temperature which varies according to the enzymes (about 45°), one observes the denaturation of the protein.

In figure 2-6, the curve in solid line is therefore the resultant of the 2 curves in dotted line: activation curve and denaturation curve. The optimal temperature is the temperature at which the 2 phenomena are in equilibrium; it depends on pH, ionic strength of the medium and also reaction time.

The shorter the reaction time, the higher could be the optimal temperature; at 50 or 60°, velocity can be very high but only for a comparatively short time, only one minute for example; (therefore, if one measures the quantity of substrate transformed — or product formed — after an interval of 10 minutes, one would erroneously find a velocity which is only one tenth of the actual velocity of the reaction during the one minute interval when the enzyme functioned before being inactivated).

Some enzymes, especially those of comparatively lower molecular weight and simple structure, stabilized by disulphide bridges less sensitive to tempera­ture rise (myokinase, ribonuclease), are specially heat resistant and can even be heated to the boiling temperature of water.

In general, enzymes seem to be less sensitive to heat in presence of the corresponding substrate (there is certainly a stabilization of the three-dimensional structure). Certain thermophile microorganisms live in water at 70 or 80°, their enzymes therefore function at those temperatures. A mutation can impart to an enzyme a thermosensitivity different from that of the enzyme of the wild strain.

2. Effect of pH:

pH variations can act on:

1. The enzyme, by modifying the degree of ionization of certain functional groups whose charge — positive or negative — is necessary, either for the formation of the enzyme-substrate complex (these are groups belonging to amino acids which constitute the “active site” or “catalytic site” of the enzyme), or for maintaining the native three-dimensional conformation of the enzymatic protein (these are groups possibly belonging to amino acids located outside the active site, at various places of the molecule);

2. The substrate, by changing its degree of ionization, which can lead to, or on the contrary prevent, the formation of the enzyme-substrate complex if the substrate must be in a given ionized form to be able to bind to the active site of the enzyme (in other words, in this case, the real substrate of the enzyme is present only in a suitable pH interval).

One can therefore define an optimum pH for the enzymatic transformation of a substrate in a medium of given composition. As shown by figure 2-7, on either side of this optimum pH, the reaction velocity decreases rapidly and, in general, at two pH units on either side of the optimum pH, velocity is negli­gible (at least 100 times less).

In the case of the pancreatic ribonuclease, the velocity is already very low at 0.5 pH unit on either side of the optimum pH, which shows the importance of a properly buffered reaction mixture for in vitro study of enzymatic reac­tions.

In vivo also, pH variations can influence the action of enzymes. In higher animals for example, the pH of blood is maintained within very nar­row limits thanks to buffer systems and to the elimination of excess acids or bases.

The optimum pH varies widely according to the enzymes. It can be highly acidic (between 1.5 and 2 for pepsin) or highly basic (between 9.5 and 10 for arginase), but it is mostly near neutrality (between 6 and 8).

Very often K_m or V_max depend on the pH in a manner resembling a titration curve; this allows the determination of the nature of an amino acid participa­ting in the formation of the E—S complex, from the pK of the ionizable group apparently implied in the interaction: a pK of 4-4.5 corresponds to a carboxylic group; a pK of 6.5—7, to an imidazole group (side chain of histidine); a pK of 8.5-9, to a – SH group; a pK of about 10.5, to a phenol group (side chain of tyrosine) or a ε-amino group (side chain of lysine).

However, certain residues have rather similar pK values, and besides, these values can be modified when the amino acid is involved in a protein (by the general charge of the protein, the presence of a nearby ionized group, a masking due to steric effects, etc.), so that the nature of the amino acid must then be confirmed by other, more rigorous methods.

3. Inhibitors:

In this study we will consider only specific inhibition, but it may be recalled that certain agents cause the denaturation or even degradation of proteins. In most cases, this is a sudden, irreversible, non-specific, phenomenon, the study of which gives no information on the mechanisms of enzymatic action.

On the contrary, the use of certain inhibitors enabled, in various cases, the determination of the nature of amino acids belonging to the active site and participating in the formation of the E — S complex.

Among substances which can bind to various groups of proteins (hydroxyl, sulphydril, carboxyl, etc.) and cause a loss of catalytic properties, either by modifying the conformation or blocking the active site of the enzyme, one finds ions of heavy metals, or compounds like mono-iodoacetic acid or para-chloromercuribenzoic acid which act on the thiol groups:

One can also cite di-isopropylfluorophosphate which enables the phos­phorylation of the serine residue of the active centre of various enzymes (esterases and certain proteolytic enzymes like trypsin) thus causing the irre­versible inactivation of the enzyme (see fig. 2-8).

Among the specific inhibitors (of an enzyme or group of enzymes) a distinc­tion is generally made between inhibitors having a competitive action and those acting in a non-competitive manner.

A. Competitive Inhibitors:

These are compounds which present a structural analogy with the substrate of the enzyme and can thus compete with it in order to bind to the active site of the enzyme.

The enzyme can therefore combine either with the substrate or with the inhibitor, and the following equilibria may be established:

It is clear that the enzyme involved in the E — I complex cannot function as catalyst; only the E —S complex will allow the formation of the reaction products. Inhibition, in other words the proportion of enzyme molecules combining with the inhibitor, depends on substrate concentration, inhibitor concentration, affinity of the enzyme for the substrate and affinity of the enzyme for the inhibitor.

The addition of a competitive inhibitor favours the dissociation of the E — S complex and therefore increases the Michaelis constant, K_m (which means that 1/K_m, affinity of the enzyme for the substrate, decreases, as shown by figure 2-9).

By adding large quantities of the substrate, the inhibitor can be displaced from the active sites it occupies. If 1/v is plotted as a function of 1/[S] as proposed by Lineweaver and Burk the resulting straight line intersects the straight line obtained in the absence of inhibitor at an infinitely high substrate concentration, i.e. when 1/[S] = 0, in other words on the vertical axis (see fig. 2-9); this is characteristic of a competitive inhibition.

Competitive inhibition exists for all enzymes. A non-metabolizable struc­tural analogue of the substrate is generally a competitive inhibitor. One may cite the example of succinodehydrogenase (see fig. 2-10) inhibited by various structural analogues of succinic acid (which differ from it only by the number of — CH₂ — between the 2 carboxylic groups).

Numerous practical applications are based on competitive inhibition, par­ticularly in therapeutics: fight against microbes, control of weeds, parasite insects etc.

The basic idea is to try to specifically inhibit an enzymatic reaction which is of capital importance in a given organism (the one being fought), but is not so in the neighbouring organisms (the host for example), either because the reaction product is dispensable in the host, or because the host can produce it by a different process.

A classical example is that of sulphamides, analogues of para-amino-benzoic acid (see fig. 2-11), a compound indispensable to many bacteria (but not to man) for the synthesis of folic acid, the coenzyme necessary for the transport of one-carbon units.

The sulphamides will compete with para-aminobenzoic acid for the active site of a bacterial enzyme catalyzing the transformation of this derivative into folic acid and this explains their bacteriostatic action.

Chemotherapy against cancer is based on numerous antimetabolites, which are generally structural analogues of purine or pyrimidine bases (constituents of nucleic acids) inhibiting the biosynthesis of nucleic acids and therefore blocking cell division (this effect is particularly pronounced in the tumoral tissues which grow much faster, but unfortunately, it manifests itself also in healthy cells).

B. Non-Competitive Inhibitors:

They bind either to the enzyme (but to a site other than the active site, so that there is no competition with the substrate for this site), or to the E — S complex to form an ESI complex, or to both. But the inhibitor is not displaced when substrate concentration is increased. Non-competitive inhibition depends on inhibitor concentration and affinity of the enzyme for the inhibitor.

In the plot of 1/v as a function of 1/[S] (see fig. 2-9), it is observed that in this case, the K_m is not modified (the straight line corresponding to non-competitive inhibition intersects the x-axis at the same point as the straight line obtained in the absence of inhibitor, therefore 1/K_m is unchanged,); on the contrary it is observed that 1/V_max is increased, therefore V_max is decreased (velocity can even become zero for total inhibition).

4. Allosteric Effectors:

Allosteric enzymes differ from other enzymes in that the plot of v as a function of [S], obtained when studying the kinetics of the reactions catalyzed by allosteric enzymes, does not give a branch of an equilateral hyperbola corresponding to the Michaelis-Menten equation (see fig. 2-4), but a sigmoid curve (S-shaped) (see fig. 2-12).

We have already come across such a curve while studying the hemoglobin ←→ oxyhemoglobin interconversion. In the case of allosteric enzymes it also reflects a cooperative effect, i.e. the fact that the binding of the first substrate molecule facilitates the binding of the second.

Allosteric enzymes have an oligomeric quaternary structure result­ing from the assembly of a variable number of protomers joined by non- covalent bonds. These oligomeric structures bear a close resemblance with that of hemoglobin.

In addition to the catalytic site where the substrate binds, these enzymes have one or several allosteric sites, which can be located on a different polypep­tide chain, and where allosteric effectors (activators or inhibitors) having no structural analogy with the substrate, can bind.

A. Allosteric Activators:

When an allosteric activator binds to the allosteric site, there results a slight modification of the conformation of the enzyme — called allosteric transition — (reversible), which causes a change of conformation of the active (catalytic) site. This site acquires a conformation more favourable to the binding of the substrate; the affinity of the enzyme for the substrate increases (K_m‘ < K_m).

Even the shape of the curve can change from the sigmoid form to the hyperbolic form (see fig. 2-12). As shown by figure 2-13, one can represent schematically, the allosteric transition caused by the binding of the activator and its effects on the active site (this scheme does not claim to represent the real structure of the enzyme; it does not show the various protomers).

B. Allosteric Inhibitors:

When an allosteric inhibitor binds to the allosteric site, an allosteric transi­tion (also reversible) takes place causing a change in the active site which now takes a conformation less favourable to the binding of the substrate (see fig. 2-14).

If v is plotted as a function of [S], the curve is again a sigmoid but a decrease of the affinity of the enzyme for the substrate is observed (K_mⁿ > K_m). In fact, this is only one of the various possible mechanisms because there are several types of inhibitions.

Very often, the enzyme catalyzing the first reaction of a metabolic pathway is an allosteric enzyme whose activity can be controlled by various allosteric effectors (activators and inhibitors). Frequently, one of the terminal products of the metabolic pathway (H in the diagram of figure 2-15) is an allosteric inhibitor of this first enzyme (E₁).

This feedback inhibition offers the very great advantage of inhibiting the first enzyme — as soon as there is an excess of terminal product — which represents a significant economy for the cell, espe­cially in the case of a sequence of reactions requiring energy (for example, during the biosynthesis of an amino acid or a nucleotide).

Thus, aspartate transcarbamylase, first enzyme of the series of reac­tions bringing about the synthesis of pyrimidine ribonucleotides, is inhibited by a terminal product of this pathway (CTP) and the various aspects of this mode of metabolism regulation by the control of the activity of enzymes.

In the case of aspartate transcarbamylase mentioned above, it is possible to dissociate the enzyme into sub-units possessing catalytic activity (their activity can no longer be modified by allosteric effectors and the kinetics is represented by a hyperbola which conforms with the Michaelis equation) and into regulatory sub-units capable of binding the allosteric effector (but deprived of catalytic activity), which shows that catalytic and allosteric sites are not only different, but also carried by different polypeptide chains joined in the quater­nary structure of the enzyme.

5. Activation or Inhibition by Covalent Modification:

The activity of numerous enzymes is controlled, not by the formation of complexes between enzymes and regulatory molecules, but by covalent modifications of the enzyme.

Of such modifications the best known is the phos-phorylation/dephosphorylation of hydroxylated amino acids; this modification, catalyzed in one direction by a kinase, and in the other by a phosphatase, plays a very important role in numerous mechanisms, like transmission of nerve impulse or metabolism of glycogen. The transformation of an inactive proen­zyme into an active enzyme by proteolytic cleavage (or cleavages).