Enzyme-substrate encounter

Kinetic and structural characterization of the known CoADR enzymes shows the occurrence of some variation with regard to their specificity. The S. aureus CoADR, a homodimer of 49kDa subunits, has a of 11 2 pmol 1 and 1.6 0.5 pmol for CoA disulfide and NADPH, respectively. Although an initial study of this enzyme reported ifcat values in the range of 1000 s (which would have had the enzyme operating near the limiting rate constant for a diffusion-controlled enzyme—substrate encounter), " subsequent kinetic analyses have shown the turnover number to be closer to 27 The enzyme shows 17% activity in the presence of... 

Wade, R. C. (1996). Brownian dynamics simulations of enzyme-substrate encounter. Bio-chem. Soc. Trans. 24(1), 254-259. 

The action of Aspergillus oryzae a-amylase on reducing-end, and uniformly radiolabelled maltotriose through maltodecaose has been studied. The enzyme was found to hydrolyse more than a single glycosidic bond during some enzyme-substrate encounters (Scheme 4). The extent of this repetitive attack was quantitated. 

The ratio of reactive to non-reactive enzyme-substrate encounters for sweet-potato jS-amylase has been measured for the degradation of amylose (DP 250) labelled with D-P C]glucose at the non-reducing end 14.6 bonds are cleaved for each effective enzyme-substrate encounter, but only 0.405 bonds are cleaved when inactive encounters are also included. The rate constants for formation and dissociation of both reactive and non-reactive enzyme-substrate complexes were also determined. 

J. J. Sines, S. A. Allison, and J. A. McCammon, Biochemistry, 29,9403 (1990). Point Charge Distributions and Electrostatic Steering in Enzyme/Substrate Encounter Brownian Dynamics of Modified Copper/Zinc Superoxide Dismutase. 

This type of bimolecular diffusion-limited reaction is prevalent in various enzyme-substrate encounters. Brownian dynamics simulations are being used to study the encounter rate of an enzyme with a substrate, protein-protein association, and nucleic add-protein association, which are just a few of the many biochemical processes that occur on characteristic time scales of diffusion-controlled systems. This article introduces the theory behind Brownian dynamics simulations and presents representative results from various areas of application. Detailed discussion of Brownian dynamics can be found in several recent papers. " ... 

In certain enzyme-substrate encounters the reactivity of the enzyme may vary with time. This may be due to a necessary conformational change or the movement of flexible loops acting as trapdoors at the entrance of the active site. Simulation of these gated reactions can be studied implicitly and explicitly using Brownian dynamics methods. Implicitly, the dynamics of the gate can be described by the rate constants... 

The results of Brownian dynamics simulations used to investigate enzyme-substrate encounter are sumiharized in Table 1. Electrostatic steering is a recurring theme, and can be treated in detail in the simulations. The treatment of flexibility and hydrodynamics is generally less sophisticated, but important insights have been derived from Brownian dynamics simulations of peptide loops that can cover enzyme active sites. 

The binary complex ES is commonly referred to as the ES complex, the initial encounter complex, or the Michaelis complex. As described above, formation of the ES complex represents a thermodynamic equilibrium, and is hence quantifiable in terms of an equilibrium dissociation constant, Kd, or in the specific case of an enzyme-substrate complex, Ks, which is defined as the ratio of reactant and product concentrations, and also by the ratio of the rate constants kM and km (see Appendix 2) ... 

In the study described the time-dependence and reversibility of the enzyme induction was not studied. Also the enzyme substrate used as a marker for the CYP 1A2 activity was caffeine, which although frequently encountered in the target population and commonly used as a marker for CYP 1A2 activity, is not a drug with a narrow therapeutic index used by the target population. The enzyme substrate used as a marker for the CYP 3A4 activity, urinary 6- 3-hydroxy-cortisol and free cortisol, although readily amenable to inclusion in studies, is not a drug and is also known to be a relatively insensitive marker for CYP 3A4 induction. Also urinary 6- 3-hydroxy-cortisol and free cortisol does not differentiate between intestinal and liver CYP 3A4 activities. 

The less frequently encountered uncompetitive inhibition occurs when a chemical binds to the enzyme-substrate complex. The catalytic function is affected without interfering with substrate binding. The inhibitor causes a structural distortion of the active site and inactivates it (Voet and Voet 2004). This has the effect of reducing the available enzyme for the reaction, and hence reduces Vmax, and also drives the reaction (E + S ES) to the right, hence decreasing Km. 

It is not necessary to use a different enzyme for each new substrate encountered. Some enzymes, such as HLADH have very broad specificities and can accommodate wide structural variations in their substrates. Also, a broad structural range of substrates is often accessible using a very limited number of enzymes of overlapping specificities (Scheme 1). 

As a first example in this regard, it is of interest to consider a study reported by E. Clementi and collaborators (108) in 1978. In this paper, the manner of treating enzymic reactions using theoretical techniques was generalized substantially from earlier work. Since the description of enzyme-substrate interactions encounters the same kinds of difficulties as the description of drug-receptor interactions, this study may provide a good indication of new directions and capabilities that can be expected to develop further in the near future. 

In textbooks dealing with enzyme kinetics, it is customary to distinguish four types of reversible inhibitions (i) competitive (ii) noncompetitive (iii) uncompetitive and, (iv) mixed inhibition. Competitive inhibition, e.g., given by the product which retains an affinity for the active site, is very common. Non-competitive inhibition, however, is very rarely encountered, if at all. Uncompetitive inhibition, i.e. where the inhibitor binds to the enzyme-substrate complex but not to the free enzyme, occurs also quite often, as does the mixed inhibition, which is a combination of competitive and uncompetitive inhibitions. The simple Michaelis-Menten equation can still be used, but with a modified Ema, or i.e. ... 

The enzymatic resolution of (R,S)-2-ethoxycarbonyl-3,6-dihydropyran has been carried out repeatedly in a pilot plant on a 100-125 kg scale. The conditions of pH, agitation, concentration, and enzyme/substrate ratio have been further optimized, but for the most part conditions developed during the laboratory optimization studies have been found to work well. This technology is currently being used to produce ton quantities of (S)-4. It has to be pointed out that no major difficulties were encountered during the scale-up. The work-up of the product in this process, unlike in many other enzyme-catalyzed processes, is quite simple and volume efficiencies are better than some chemical reactions. The recovery and recycling of the enzyme is not needed since it is commercially available and relatively inexpensive. 

For some problems, such as the motion of heavy particles in aqueous solvent (e.g., conformational transitions of exposed amino acid sidechains, the diffusional encounter of an enzyme-substrate pair), either inertial effects are unimportant or specific details of the dynamics are not of interest e.g., the solvent damping is so large that inertial memory is lost in a very short time. The relevant approximate equation of motion that is applicable to these cases is called the Brownian equation of motion,... 

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