Single enzyme molecule kinetics

Substrates may affect enzyme kinetics either by activation or by inhibition. Substrate activation may be observed if the enzyme has two (or more) binding sites, and substrate binding at one site enhances the alfinity of the substrate for the other site(s). The result is a highly active ternary complex, consisting of the enzyme and two substrate molecules, which subsequently dissociates to generate the product. Substrate inhibition may occur in a similar way, except that the ternary complex is nonreactive. We consider first, by means of an example, inhibition by a single substrate, and second, inhibition by multiple substrates. 

Structural studies of the oxy-Cope catalytic antibody system reinforce the idea that conformational dynamics of both protein and substrate are intimately intertwined with enzyme catalysis, and consideration of these dynamics is essential for complete understanding of biologically catalyzed reactions. Indeed, recent single molecule kinetic studies of enzyme-catalyzed reactions also suggest that different conformations of proteins are associated with different catalytic rates (Xie and Lu, 1999). In addition, a number of enzymes are known to undergo conformational changes on binding of substrate (Koshland, 1987) that lead to enhanced catalysis two examples are hexokinase (Anderson and Steitz, 1975 Dela-Fuente and Sols, 1970) and triosephosphate isomerase (Knowles, 1991). 

True lipases show the interfacial activation phenomenon in their catalytic activity pattern. At low concentration of water-insoluble substrates, lipases are almost inactive, and the hydrolytic activity does not increase linearly. At a certain substrate concentration, however, the hydrolytic activity of lipases increases rapidly and the lipase kinetics resembles normal enzyme kinetics. This boost in activity is related to the formation of water-insoluble substrate aggregates such as micelles or another second phase. Only when this second phase is present, do lipases become fully active. This interfacial activation is caused by a large conformational change in the 3D structure of the lipases. In their water-soluble form, the active site is covered by a lid, which prevents the substrates from reaching it. At the lipidAvater interface, the lid is opened and the active site is accessible to the substrates. In addition, the now accessible area is mainly hydrophobic, which gives the open-form lipase the shape and behavior of conventional surfactant molecules with a hydrophilic and a hydrophobic moiety in one single molecule. 

Figure 12.9 Single moLecuLe studies can reveal enzyme kinetic information. Left the acto-myosin ATPase cycle lined up with a typical binding event the event begins when myosin binds to actin and ends when an ATP molecule diffuses into the binding cleft, causing it to dissociate from the actin filament. Right the event lifetimes of rabbit skeletal myosin-II SI fragment are stochastic and show an exponential distribution because each event is terminated by a single Poisson process (ATP binding). Note that the inset has two points removed, as zeros cannot be plotted on a log scale...

A frequent case in enzyme kinetics is the ability of enzymes to form numerous non-productive complexes with substrates, that do not break down (Fersht, 1999). If an enzyme molecule binds a single substrate molecule in a productive complex EA, but also another substrate molecule in a non-productive complex AE, we are referring to non-productive binding of this substrate. Akinetic model is analogous to that for a linear competitive inhibition (Section 5.2). 

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