Enzymes substrate dependence

Acyloins (a-hydroxy ketones) are formed enzymatically by a mechanism similar to the classical benzoin condensation. The enzymes that can catalyze reactions of this type arc thiamine dependent. In this sense, the cofactor thiamine pyrophosphate may be regarded as a natural- equivalent of the cyanide catalyst needed for the umpolung step in benzoin condensations. Thus, a suitable carbonyl compound (a -synthon) reacts with thiamine pyrophosphate to form an enzyme-substrate complex that subsequently cleaves to the corresponding a-carbanion (d1-synthon). The latter adds to a carbonyl group resulting in an a-hydroxy ketone after elimination of thiamine pyrophosphate. Stereoselectivity of the addition step (i.e., addition to the Stand Re-face of the carbonyl group, respectively) is achieved by adjustment of a preferred active center conformation. A detailed discussion of the mechanisms involved in thiamine-dependent enzymes, as well as a comparison of the structural similarities, is found in references 1 -4. 

The response characteristics of enzyme electrodes depend on many variables, and an understanding of the theoretical basis of their function would help to improve their performance. Enzymatic reactions involving a single substrate can be formulated in a general way as... 

The kinetics of enzyme reactions were first studied by the German chemists Leonor Michaelis and Maud Menten in the early part of the twentieth century. They found that, when the concentration of substrate is low, the rate of an enzyme-catalyzed reaction increases with the concentration of the substrate, as shown in the plot in Fig. 13.41. However, when the concentration of substrate is high, the reaction rate depends only on the concentration of the enzyme. In the Michaelis-Menten mechanism of enzyme reaction, the enzyme, E, and substrate, S, reach a rapid preequilibrium with the bound enzyme-substrate complex, ES ... 

Steiner RA, KH KaUc, BW Dijkstra (2002) Anaerobic enzyme substrate structures provide insight into the reaction mechanism of the copper-dependent quercitin 2,3-dioxygenase. Proc Natl Acad USA 99 16625-16630. 

The actual velocity of the reaction depends on how much of the total amount of enzyme is present in the enzyme-substrate (ES) complex. At low substrate concentrations, very little of the enzyme is present as the ES complex—most of it is free enzyme that does not have substrate bound. At very high substrate concentrations, virtually all the enzyme is... 

For endopectate lyases, the rate of cleavage of glycosidic bonds and the affinity of the enzyme for the substrate depend on the chain length,240,241 as with endo-D-galacturonanase. The frequency of splitting of bonds 2 and 3 in tetra(D-galactosiduronic acid) is different. Endopectate lyase of Bacillus polymyxa splits240 bond 3 1.4 times faster than bond 2. 

The affinity of pectin lyase for the substrate depends on its d.e. Voragen and coworkers232 observed that, for pectin lyase from a commercial preparation, 1/Km values decreased as the d.e. decreased. On the other hand, the values of V did not depend on the d.e. The effect of the d.e. on the affinity of the enzyme for the substrate was attributed to the lower content of reactive sites in the less esterified substrates. Values of 1 IKm increased with decreasing pH. Voragen and coworkers232 contended that the charged groups of the enzyme, or of the substrate, play a role in the formation of the enzyme-substrate complex. 

The kinetics of the general enzyme-catalyzed reaction (equation 10.1-1) may be simple or complex, depending upon the enzyme and substrate concentrations, the presence/absence of inhibitors and/or cofactors, and upon temperature, shear, ionic strength, and pH. The simplest form of the rate law for enzyme reactions was proposed by Henri (1902), and a mechanism was proposed by Michaelis and Menten (1913), which was later extended by Briggs and Haldane (1925). The mechanism is usually referred to as the Michaelis-Menten mechanism or model. It is a two-step mechanism, the first step being a rapid, reversible formation of an enzyme-substrate complex, ES, followed by a slow, rate-determining decomposition step to form the product and reproduce the enzyme ... 

Almost all the FDH molecules on the electrode surface seemed to retain the enzyme activity because of the mild immobilization at less extreme potential. The enzyme activity of immobilized FDH was dependent on the thickness of polypyrrole membrane because a thicker membrane could prevent the enzyme substrate from diffusing into the membrane matrix. Therefore, it was very important to make the polypyrrole membrane as thin as possible to minimize the effect on substrate diffusion and to ensure the complete coverage of the enzyme layer. 

In reversible inhibition, the inhibitor may bind or associate to enzyme or enzyme-substrate complex. Depending on the binding/combination inhibitor, the reversible inhibition may be of three types. 

The rate at which the substrate is converted to the product by the action of an enzyme is dependent upon the concentrations of both the enzyme and the substrate. The initial rate of such a reaction is maximal and it is this initial rate of reaction that reflects the enzyme activity (Figure 8.12). The major factor in this decline is the depletion of the substrate but the increasing amount of product competing for the enzyme also reduces the rate of the forward reaction. Additionally, inactivation of the enzyme may occur, particularly if an appreciable reaction time is involved. 

Thus, intramolecular activation (cyclization-elimination) in this series is modulated by steric factors. In addition, hydrolysis may be enzyme-catalyzed, depending on substrates and biological conditions. 

Related to this is the volume change associated with dipole development in transition states. This has been investigated theoretically for a model substance of molecules with a size similar to that of water (Morild and Larsen, 1978). The calculations show that the volume changes are very pressure-dependent in this case. A change in dipole moment from 0 to 1 x 10-3 C-m gives a volume decrease of about 30 cm3 mol-1 at 350 bar and about 20 cm3 mol-1 at 750 bar. However, this may not be typical for molecules as large as enzyme-substrate complexes. 

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