Precision enzyme-catalyzed reactions

Terpenes illustrate an important principle of the molecular logic of living systems—namely that in building large molecules, small subunits are strung together by an iterative process and then chemically modified by precise enzyme-catalyzed reactions. 

The structure of atoms is known to us in great detail down to the fact that isotopes are not totally equal to the major form of an element. Heavier isotopes react slower in enzyme-catalyzed reactions but they form stronger bonds. No uncertainty is needed for that aspect of our world. Heisenbergs Principle does not pertain to the power fields created by the atomic nuclear structure, but rather to the position of the electrons within these power fields. For the macroscopic world and its chemical basis the position of the power fields (orbitals) is important, and these positions allow carbon, very precisely and very predictably, to give rise to four bonds (or two double bonds as in CO2 or a triple and a single bond as in cyanate) that are stable under a variety of conditions and which allow carbon to form the many and varied polymers that form the skeleton of life. Chemistry does not suffer uncertainty neuroses. 

Some enzymatic reactions can be followed directly, either by substrate depletion or product accumulation measurements, with adequate precision for direct enzymatic assays. However, many enzymes catalyze reactions involving species that are not themselves readily measured. In these situations, products are converted to species that are measurable, in a coupled, or indicator reaction. The indicator reaction may be chemical or enzymatic, and quantitatively converts the product of the primary reaction into a readily measurable species. The main requirement for the indicator reaction, whether it is chemical or enzymatic in nature, is that the conversion of the primary product into the measured product must be rapid and quantitative. 

How does enzyme-substrate binding result in a faster chemical reaction The precise answer to this question is probably different for each enz)une-substrate pair, and, indeed, we understand the exact mechanism of catalysis for very few enzymes. Nonetheless, we can look at the general features of enz)une-substrate interactions that result in enhanced reaction rate and product formation. To do this, we must once again look at the steps of an enzyme-catalyzed reaction, focusing on the remaining steps highlighted in blue ... 

Formation of an enzyme-substrate complex is the first step of an enzyme-catalyzed reaction. This involves the binding of the substrate to the active site of the enzyme. The lock-and-key model of substrate binding describes the enzyme as a rigid structure into which the substrate fits precisely. The newer induced fit model describes the enzyme as a flexible molecule. The shape of the active site approximates the shape of the substrate and then "molds" itself around the substrate. 

This is to say that enzymes evolve by maximization of this ratio approaching the diffusion-control limit, which is obviously dependent on the equilibrium constant of the catalyzed reaction. Arguably this reasoning is only valid for conditions of initial rates, which are not the typical situation in living cells, where an enzyme is embedded in a chain of reactions with no initial velocities and thus, the driving force of natural selection could be contemplated as reaching the precise kcaJK value to maximize the conversion of substrate fluxes. Table 2 summarizes the most important parameters that define the kinetics of a monomolecular enzyme-catalyzed reaction. 

Virtually all enzymatic assays are carried out at 20-50 °C in aqueous buffers of known pH and controlled composition. Both temperature and buffer properties affect the rates of enzyme- catalyzed reactions markedly. The effects of temperature can usually be summarized by a bell-shaped curve (Fig. 4 A). At lower temperatures, reaction rates increase with temperature, but beyond a certain point, denaturation (unfolding) of the enzyme molecules begins, so they lose their ability to bind the substrate, and the reaction rate falls. The temperature giving maximum activity varies from one enzyme to another, according to the robustness of the molecule. In some cases, it may be convenient to use a temperature rather below this maximum, otherwise the rate becomes too high to measure precisely. The rates of many enzyme-catalyzed reactions increase by a factor of ca. 2 over a range of I0°C in the region below the maximum of the... 

Since most synthetic applications require enzymes catalyzing nonnatural substrates, their properties often have to be improved. One way to achieve this is to optimize reaction conditions such as pH, temperature, solvents, additives, etc. [6-9]. Another way is to modulate the substrates without compromising the synthetic efficiency of the overall reaction [10]. In most cases for commercial manufacturing, however, the protein sequences have to be altered to enhance reactivity, stereoselectivity and stability. It was estimated that over 30 commercial enzymes worldwide have been engineered for industrial applications [11]. Precise prediction of which amino acids to mutate is difficult to achieve. Since the mid 1990s, directed evolution... 

Carbon kinetic isotope effects on enzyme-catalyzed decarboxylations are among the most intensively studied enzyme reactions. This is because of the central role that carbon dioxide plays in plant metabolism and also because precise kinetic measurements are relatively easy to obtain since the carbon dioxide liberated in the reaction can be immediately analyzed using isotope ratio mass spectrometry. 

The catalytic action of an enzyme, its activity, is measured by determining the increase in the reaction rate under precisely defined conditions—i.e., the difference between the turnover (violet) of the catalyzed reaction (orange) and uncatalyzed reaction (yellow) in a specific time interval. Normally, reaction rates are expressed as the change in concentration per unit of time (mol 1 s see p. 22). Since the catalytic activity of an enzyme is independent of the volume, the unit used for enzymes is usually turnover per unit time, expressed in katal (kat, mol s ). However, the international unit U is still more commonly used (pmol turnover min 1 U = 16.7 nkat). 

Protein function can be described on three levels. Phenotypic function describes the effects of a protein on the entire organism. For example, the loss of the protein may lead to slower growth of the organism, an altered development pattern, or even death. Cellular function is a description of the network of interactions engaged in by a protein at the cellular level. Interactions with other proteins in the cell can help define the lands of metabolic processes in which the protein participates. Finally, molecular function refers to the precise biochemical activity of a protein, including details such as the reactions an enzyme catalyzes or the ligands a receptor binds. 

Next, the mechanism of the Type II reactions is discussed. To discriminate one of the enantiofaces of the acceptor it is desirable to place and to activate the electrophiles in a chiral environment. At the same time, effective activation of the Michael donor is required. In Shibasaki s ALB-catalyzed reaction (Scheme 3), it was proposed that the aluminum cation functioned as a Lewis acid to activate enones at the center of the catalyst, and that the Li-naphthoxide moiety deproton-ated the a-hydrogen of malonate to form the Li enolate (Scheme 9). Such simultaneous activation of both reactants at precisely defined positions became feasible by using multifunctional heterobimetallic complexes the mechanism is reminiscent of that which is operative in the active sites of enzymes. The observed absolute stereochemistry can be understood in terms of the proposed transition state model 19. Importantly, addition of a catalytic amount of KOt-Bu (0.9equiv. to ALB) was effective in acceleration of the reaction rate with no deterioration of the... 

We chose this reaction since it has been proposed as a model for the rearrangement of 2-methyleneglutarate to 3-methylitaconate, catalyzed by the coenzyme-B,2-dependent enzyme, 2-methyleneglutarate mutase [16, 26, 57]. More specifically, equation 2 represents the second step in the addition-elimination pathway (reaction c. Scheme 4) for a 1,2-shift. Additionally, this reaction has been widely studied experimentally [58] and has been described as the most precisely calibrated radical reaction [59]. 

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