enzyme_kinetics2

enzyme_kinetics2 - Enzyme Kinetics Part B Multisubstrate...

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Enzyme Kinetics: Part B Multisubstrate Enzyme Kinetics So far, we have considered only single substrate enzyme kinetics. Although this is common, equally common are enzymes that use two substrates. Less frequently, enzymes use three substrates, but we will not consider this. It is important to make clear at this point the distinctions between three different levels of mechanism. The first level is the kinetic mechanism . This is the order (or lack of) in which substrates bind and react with the enzyme. This level is relevant, of course, only to multisubstrate systems since there is only one way to bind a single substrate. The second level is the chemical mechanism. This concerns the elucidation of the chemical structures of all intermediates occurring in the transformation of substrates to products. The third level of mechanism is the catalytic mechanism . Here, one is concerned with figuring out exactly what tricks the enzyme uses to make chemical reactions on the enzyme surface occur much faster than they do in solution. Enzymes using two substrates can be classified into two overall groups of kinetic mechanisms: 1) sequential and 2) ping-pong. The two groups are fundamentally different. The name "sequential" means that both substrates must bind in sequence (either an ordered or a random sequence) before reaction can occur . This means that both substrates are present at the active site and the enzyme catalyzes a reaction between the two substrates directly . This is not the case for ping-pong mechanisms (see below), and this is the fundamental difference between the two mechanistic groups. Sequential mechanisms can be either random or ordered. "Random" and "ordered" refer to the timing in which substrates bind.
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2 Random sequential: E EA EB EAB E + P + Q A B B A k cat Ordered sequential: E A EA B EAB k cat E + P + Q In a random sequential mechanism, either substrate can bind first. This usually happens when the two substrate binding sites both have unencumbered access to solvent, represented schematically below. A B A B Random Ordered Ordered binding occurs when the B substrate binding site is "on top" of the A binding site. Here, A physically cannot get on or off the enzyme when B is bound; to form a productive EAB complex A must bind first.
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3 Ping-pong mechanisms were so named because the enzyme bops back and forth between two different forms like a ping-pong ball goes back and forth from one side of the table to the other. E EA E' E'B A B P Q This kinetic mechanism is composed of two half-reactions. First, A reacts with E all by itself to give E' and P. E' is a modified enzyme form. Usually one part of A is put onto E to give E'. For example, the modification of E to give E' could be a phosphorylation as in protein tyrosine phosphatases. Both substrates generally use the same binding site in a ping-
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enzyme_kinetics2 - Enzyme Kinetics Part B Multisubstrate...

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