KINETICS OF MULTI SUBSTRATE ENZYME CATALYZED REACTION

 Enzyme kinetics is the study of the chemical reactions that are catalyzed by enzymes. Enzymes are usually protein molecules that manipulate other molecules, the enzyme’s substrates. These target molecules bind to an enzyme’s active site and are transformed into products through a series of steps known as the enzyme mechanism.

These mechanisms can be divided into: Single substrate mechanism Multiple- substrate mechanism. An essential feature of enzyme-catalyzed reactions is saturation: at increasing concentrations of substrates the rate increases and approaches a limit where there is no dependence of rate on concentration this is known as Vmax.

Multi-substrate mechanism

Involve more than one substrate often resulting in multiple products known as bi bi reactions (two substrates two products one enzyme). A and B are the substrates, E is the enzyme and P, Q are the products (considering the forward direction).

Multi- substrate mechanism is divided into sequential mechanisms and non- sequential mechanisms.

1. In the sequential type, both substrates must bind to the enzyme before any product is released. The sequential mechanism can be subdivided int0: random-order mechanisms, in which the order of binding the substrates is not important, and compulsory-order mechanisms, when the order of binding is important.

Sequential bi-bi

• The first important type of bi-bi reaction is known as sequential, which means that all substrates must add to the enzyme before any reaction takes place
• The sequential bi-bi can be
  • random, any substrate can bind first to the enzyme and any product can leave first
  • ordered, meaning that the substrates add to and products leave the enzyme in a specific order .

Sucrose                fructose                Pi                G-1-P

E                                                                                       E

       E.sucrose    E.glucose.fructose      E-glucose         E.glucose-1-P    

B. Non-sequential mechanisms on the other hand, do not require all the substrates to bind before a product is released and thus a substituted- enzyme intermediate, E* will be formed. Such mechanisms are generally referred to as ping-pong or substituted-enzyme mechanisms.

Ping-pong mechanism, also called a double-displacement reaction, is characterized by the change of the enzyme into an intermediate form when the first substrate to product reaction occurs. It is important to note the term intermediate indicating that this form is only temporary. At the end of the reaction the enzyme must be found in its original form.

Enzymes with a ping-pong mechanism can exist in two states, E and a chemically modified form of the enzyme E*; this modified enzyme is known as an intermediate. In such mechanisms, substrate A binds, changes the enzyme to E* by, for example, transferring a chemical group to the active site, and is then released. Only after the first substrate is released can substrate B bind and react with the modified enzyme, regenerating the unmodified E form and product Q is released. Examples of enzymes that work through ping pong mechanism include some oxidoreductases such as peroxidase, and some transferases etc.

 In further explanation of the Ping-pong bi-bi (double-displacement) reaction;

• One substrate bind first to the enzyme followed by product P release
• Typically, product P is a fragment of the original substrate A
• The rest of the substrate is covalently attached to the enzyme E, which we now designate as F
• Now the second reactant, B, binds and reacts with the enzyme to form a covalent adduct with the covalent fragment of A still attached to the enzyme to form product Q
• This is now released and the enzyme is restored to its initial form, E

Enzyme complexes

The form of an enzyme that exists in solution in the absence of any substrate or other small molecule that can bind to it is called the free enzyme. An intermediate derived from the free enzyme by binding of a substrate molecule is called an enzyme-substrate complex, and terms such as enzyme-product complex, enzyme-inhibitor complex. A complex derived from the free enzyme and one other molecule is called a binary complex; one derived from the free enzyme and two other molecules is called a ternary complex. A ternary complex (E + both substrates) is formed in both cases. In these enzymes, both substrates bind to the enzyme at the same time to produce an EAB ternary complex.