Enzyme-substrate studies

Kinetic studies involving enzymes can principally be classified into steady and transient state kinetics. In tlie former, tlie enzyme concentration is much lower tlian that of tlie substrate in tlie latter much higher enzyme concentration is used to allow detection of reaction intennediates. In steady state kinetics, the high efficiency of enzymes as a catalyst implies that very low concentrations are adequate to enable reactions to proceed at measurable rates (i.e., reaction times of a few seconds or more). Typical enzyme concentrations are in the range of 10 M to 10 ], while substrate concentrations usually exceed lO M. Consequently, tlie concentrations of enzyme-substrate intermediates are low witli respect to tlie total substrate (reactant) concentrations, even when tlie enzyme is fully saturated. The reaction is considered to be in a steady state after a very short induction period, which greatly simplifies the rate laws. 

Pioneering enzyme specificity studies at the turn of the century by the great organic chemist Emil Eischer led to the notion of an enzyme resembling a lock and its particular substrate the key. This analogy captures the essence of the specificity that exists between an enzyme and its substrate, but enzymes are not rigid templates like locks. 

The concept of electrostatic complimentarity is somewhat meaningless without the ability to estimate its contribution to AAg. Thus, it is quite significant that the electrostatic contribution to AAthat should be evaluated by rigorous FEP methods can be estimated with a given enzyme-substrate structure by rather simple electrostatic models (e.g., the PDLD model). It is also significant that calculated electrostatic contributions to A A g seem to account for its observed value (at least for the enzymes studied in this book). This indicates that simple calculations of electrostatic free energy can provide the correlation between structure and catalytic activity (Ref. 10). 

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 ... 

Nanninga, L.B. Mommaerts. C.R. (1960). Studies on the formation of an enzyme substrate complex between myosin and adenosine-triphosphate. Proc. Natl. Acad. Sci. USA 46, 1155-1166. 

The successful use of these X-ray crysallographic techniques in studying the enzyme-substrate interactions of lysozyme (21) and chymotrypsin (22) has recently been reviewed by Blow and Steitz (16) and Blow (23). To date, however, these methods have had only limited application, since the detailed structures of only about ten enzymes have been elucidated by X-ray diffraction... 

Substrate specificity and mode of action. Previous information, which we had obtained from FORL crude culture filtrates, showed that the pectin lyase (characterized by an isoelectric point of 9.2) had a predominantly "endo" way of action. This fact has been confirmed with the purified protein it decreased the viscosity of reaction mixtures with pectin, but no increase in absorbance was detected in standard conditions. Moreover, the enzyme showed a great specificity for the substrate, as no activity was detected when the decrease in viscosity of pectate was tried. So, properties of the purified enzyme were studied by using pectin as substrate and following the decrease in viscosity of the reaction mixtures. 

Maximal speed (Vmax) and supposed Michaelis constant (K ) of pectin hydrolysis reaction (catalyzed by the studied pectinesterase) were determined in Zinewedwer — Berk coordinated, They were determined in the range of substrate concentration values that was below optimum one V = 14.7 10 M min K = 5.56 10 M. The value of dissociated constant (KJ of the triple enzyme—substrate complex was determined from the experimental data at high substrate concentration. It was the following Kj= 0.22 M. Bunting and Murphy method was used for determination. 

Since many years, pectolytic enzymes have been widely used in industrial beverage processing to improve either the quality and the yields in fruit juice extraction or the characteristics of the final product [1,2]. To this purpose, complex enzymatic mixtures, containing several pectolytic enzymes and often also cellulose, hemicellulose and ligninolytic activities, are usually employed in the free form. The interactions among enzymes, substrates and other components of fruit juice make the system very difficult to be investigated and only few publications are devoted to the study of enzymatic pools [3-5], An effective alternative way to carry out the depectinisation process is represented by the use of immobilized enzymes. This approach allows for a facile and efficient enzymatic reaction control to be achieved. In fact, it is possible to avoid or at least to reduce the level of extraneous substances originating from the raw pectinases in the final product. In addition, continuous processes can be set up. 

Ethanolamine ammonia lyase has a molecular weight of 520,000 and consists of 8 or 10 subunits. Two 5 -deoxyadenosylcobalamin molecular bind per enzyme molecule, and recent kinetic studies by Babior show that these two molecules carry out catalysis independently. Evidence is available that this enzyme functions by a radical mechanism since both spin labeling and Co(II) esr experiments indicate that Co(II) is an intermediate during H-transfer. Also, 5 -deoxyadenosine has been detected as a product of oxygenation of the enzyme-substrate complex (99—101). 

A second enzyme, BC02, was identified that cleaves carotenoids asymmetrically at the 9,10-double bond to produce the 10-apocarotenal (C27) and (3-ionone (C13), in a reaction similar to the Arabidopsis CCD7. Examples of BC02 have been cloned from mouse, zebra fish, ferret, and human (Kiefer et al. 2001, von Lintig et al. 2005, Hu et al. 2006). Substrate studies with different BC02s showed that these enzymes prefer acyclic carotenoids such as lycopene over cyclic carotenoids (Kiefer et al. 2001, von Lintig et al. 2005, Hu et al. 2006). These enzymes also seem to be selective for different carotenoid isomers. BC02 from ferret for example cleaves d,v-isomers of lycopene but not all-trans-lycopene (Hu et al. 2006). 

In an unpublished study, Grieger and Hansel have used absorbance measurements to monitor the progress of the reaction forming an enzyme-substrate complex of imidazole (S) and metmyo-globin (E) at high pressures. Formation of the enzyme-substrate complex (ES) may be represented by the following equation... 

In kinetic studies of enzymatic reactions, rate data are usually tested to determine if the reaction follows the Michaelis-Menten model of enzyme-substrate interaction. Weetall and Havewala [Biotechnol. and Bioeng. Symposium 3 (241), 1972] have studied the production of dextrose from cornstarch using conventional... 

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