Structural Biochemistry/Enzyme/Multi-substrate reactions
When dealing with enzyme substrate reactions, most involve a single substrate which is turned into a single product by an enzyme. However, for multi-substrate reactions there are more than one substrate involved. The reaction involves a complex reaction that not only tells where the substrates bind, but the sequence of binding as well. For instance if there were two substrates, one would be labeled substrate A and substrate B. On the basis that one of the substrate concentrations remains constant the entire time like substrate A, the enzyme would behave in the same manner as a single substrate enzyme. Because of this, the a graph of substrate concentration over velocity would produce values of both Km and Vmax for substrate B because substrate A is constant. The value of Km represents the substrate concentration at which the reaction is half its maximum velocity (Vmax). However, this is not always the case and when the concentrations of substrate A and substrate B are different the result of the enzyme-substrate interactions can be explained by two different mechanisms that will be described below. Furthermore, hypothetically, substrate A and substrate B would lead to two different products that can be labeled as P and R respectively.
B. Ternary Complex Mechanism
In a Ternary Complex Mechanism two substrates bind to the enzyme (hypothetically substrate A and substrate B) to form a complex that is known as the EAB Ternary Complex. The order of the substrate binding can either be in a specific sequence (ordered) or random sequence as well. An enzyme that follows the ternary complex mechanism would have a Lineweaver-Burk Plot that has two lines that intersect on a reciprocal substrate concentration/velocity graph. The plot represents a linear graph of the reciprocals 1/S and 1/V. A specific enzyme that has a ternary-complex mechanism is DNA polymerase. DNA polymerase functions to add nucleotides to DNA.
C. Ping Pong Mechanism
For this mechanism, an enzyme can be in two states. One of the states is labeled E and the other state that is also known as the intermediate and that is chemically modified is labeled E*. In this mechanism, the first substrate (substrate A) binds to enzyme turning it into E* by the transfer of a chemical group to the active site and then the substrate is released. Once substrate A is released, substrate is able to bind to the modified Enzyme (E*) forming the unmodified Enzyme once again (regeneration). When a Line-Weaver Burk plot is graphed, two sets of parallel will be formed opposite of the Ternary Complex Mechanism. Specific enzymes that follow this mechanism include oxidoreductases and serine proteases. Some of the serine proteases include the digestive enzymes of chymotrypsin and trypsin. For the example of the chymotrypsin, an acyl-enzyme is formed after the breakdown of the tetrahedral intermediate, which is formed after the nucleophilic attack of Ser to the carbonyl forming the intermediate. Once the intermediate breaks down, the acyl-enzyme is formed which acts as the modified Enzyme (E*). The acyl-enzyme however breaks down later into the intermediate complex as the amine group of the acyl-enzyme (E*) leaves and hydrogen functions as a nucleophile to attack the carbonyl forming the tetrahedral intermediate once again.