Enzymes barriers

Van Gelder NM. A possible enzyme barrier for y-aminobutyric acid in the central nervous system. Prog Brain Res 1967 29 259-268. 

The purpose of this chapter is to document the nature of the enzyme barrier that macromolecular drugs will encounter during their passage down the human gastrointestinal tract. I will discuss the peptidases that digest peptides and proteins and the nucleases that will hydrolyse nucleic acids and also briefly consider other enzymes that may affect the behaviour of the newer generation of pharmaceutical formulations. It is very important to consider both the qualitative aspects of the problem, that is, the specificity of the digestive... 

A. (The gas phase estimate is about 100 picoseconds for A at 1 atm pressure.) This suggests tliat tire great majority of fast bimolecular processes, e.g., ionic associations, acid-base reactions, metal complexations and ligand-enzyme binding reactions, as well as many slower reactions that are rate limited by a transition state barrier can be conveniently studied with fast transient metliods. 

Although extraction of lipids from membranes can be induced in atomic force apparatus (Leckband et al., 1994) and biomembrane force probe (Evans et al., 1991) experiments, spontaneous dissociation of a lipid from a membrane occurs very rarely because it involves an energy barrier of about 20 kcal/mol (Cevc and Marsh, 1987). However, lipids are known to be extracted from membranes by various enzymes. One such enzyme is phospholipase A2 (PLA2), which complexes with membrane surfaces, destabilizes a phospholipid, extracts it from the membrane, and catalyzes the hydrolysis reaction of the srir2-acyl chain of the lipid, producing lysophospholipids and fatty acids (Slotboom et al., 1982 Dennis, 1983 Jain et al., 1995). SMD simulations were employed to investigate the extraction of a lipid molecule from a DLPE monolayer by human synovial PLA2 (see Eig. 6b), and to compare this process to the extraction of a lipid from a lipid monolayer into the aqueous phase (Stepaniants et al., 1997). 

Cell membrane The cell membrane is composed of about 45% lipid and 55% protein. The lipids form a bilayer that is a continuous nonpolar hydrophobic phase in which the proteins are embedded. The cell membrane is a highly selective permeability barrier that controls the entry of most substances into the cell. Important enzymes in the generation of cellular energy are located in the membrane. 

A chemical reaction that will occur spontaneously because the energy level of the products (P) is less than the energy level of the substrate (S). (a) In the absence of an enzyme, acfivafion energy is high. Few molecules have sufficient energy to overcome this barrier and the reaction proceeds slowly if at all. (b) In the presence of an enzyme, acfivafion energy is lower and the reaction proceeds more quickly. 

However, there are disadvantages to using immobilised cells. The cell may contain numerous catalytically active enzymes, which may catalyse unwanted side reactions. Also, the cell membrane itself may serve as a diffusion barrier, and may reduce productivity. The matrix may sharply reduce productivity if the microorganism is sensitive to product inhibition. One of the disadvantages of immobilised cell reactors is that the physiological state of the microorganism cannot be controlled. 

Further improvements can be achieved by replacing the oxygen with a non-physiological (synthetic) electron acceptor, which is able to shuttle electrons from the flavin redox center of the enzyme to the surface of the working electrode. Glucose oxidase (and other oxidoreductase enzymes) do not directly transfer electrons to conventional electrodes because their redox center is surroimded by a thick protein layer. This insulating shell introduces a spatial separation of the electron donor-acceptor pair, and hence an intrinsic barrier to direct electron transfer, in accordance with the distance dependence of the electron transfer rate (11) ... 

Electroosmotic flow, 195 End column detection, 89 Energy barrier, 16 Enzyme electrodes, 172, 174 Enzyme immunoassays, 185 Enzyme inhibition, 181 Enzyme reconstitution, 178 Enzyme wiring, 178 Equilibrium potential, 15 Ethanol electrodes, 87, 178 Exchange current, 14... 

FIGURE 5.8. A downhill trajectory for the proton transfer step in the catalytic reaction of trypsin. The trajectory moves on the actual ground state potential, from the top of the barrier to the relaxed enzyme-substrate complex. 1, 2, and 3 designate different points along the trajectory, whose respective configurations are depicted in the upper part of the figure. The time reversal of this trajectory corresponds to a very rare fluctuation that leads to a proton transfer from Ser 195 to His 57. 

The overall catalytic rate constant of SNase is (see, for example, Ref. 3) kcat — 95s 1 at T = 297K, corresponding to a total free energy barrier of Ag at = 14.9 kcal/mol. This should be compared to the pseudo-first-order rate constant for nonenzymatic hydrolysis of a phosphodiester bond (with a water molecule as the attacking nucleophile) which is 2 x 10 14 s corresponding to Ag = 36 kcal/mol. The rate increase accomplished by the enzyme is thus 101S-1016, which is quite impressive. 

Once the energetics of the reference reaction are estimated we are ready to analyse the effect of the enzyme, which reduces the barrier from —25 kcal/mol to —9 kcal/mol, with the first step (H20— H+ + OH-) as the... 

The exercise given above should overestimate the activation barrier in the enzyme, since it does not take into account the secondary transfer of the proton from water to histidine. A more complete study (Fig. 8.8) that... 

FIGURE 9.1. The potential surface for proton transfer reaction and the effect of constrainir the tiA B distance. The figure demonstrates that the barrier for proton transfer increasi drastically if the A — B distance is kept at a distance larger than 3.5 A. However, in solutic and good enzymes the transfer occurs through pathway a where the A - B distance is arour 2.7 A. 

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