Transition State Theory - Applications of TST: Enzymatic Reactions

Applications of TST: Enzymatic Reactions

Enzymes catalyze chemical reactions at rates that are astounding relative to uncatalyzed chemistry at the same reaction conditions. Each catalytic event requires a minimum of three or often more steps, all of which occur within the few milliseconds that characterize typical enzymatic reactions. According to transition state theory, the smallest fraction of the catalytic cycle is spent in the most important step, that of the transition state. The original proposals of absolute reaction rate theory for chemical reactions defined the transition state as a distinct species in the reaction coordinate that determined the absolute reaction rate. Soon thereafter, Linus Pauling proposed that the powerful catalytic action of enzymes could be explained by specific tight binding to the transition state species Because reaction rate is proportional to the fraction of the reactant in the transition state complex, the enzyme was proposed to increase the concentration of the reactive species.

This proposal was formalized by Wolfenden and coworkers at University of North Carolina at Chapel Hill, who hypothesized that the rate increase imposed by enzymes is proportional to the affinity of the enzyme for the transition state structure relative to the Michaelis complex. Because enzymes typically increase the non-catalyzed reaction rate by factors of 1010-1015, and Michaelis complexes often have dissociation constants in the range of 10−3-10−6 M, it is proposed that transition state complexes are bound with dissociation constants in the range of 10-14 -10−23 M. As substrate progresses from the Michaelis complex to product, chemistry occurs by enzyme-induced changes in electron distribution in the substrate.

Enzymes alter the electronic structure by protonation, proton abstraction, electron transfer, geometric distortion, hydrophobic partitioning, and interaction with Lewis acids and bases. These are accomplished by sequential protein and substrate conformational changes. When a combination of individually weak forces are brought to bear on the substrate, the summation of the individual energies results in large forces capable of relocating bonding electrons to cause bond-breaking and bond-making. Analogs that resemble the transition state structures should therefore provide the most powerful noncovalent inhibitors known, even if only a small fraction of the transition state energy is captured.

All chemical transformations pass through an unstable structure called the transition state, which is poised between the chemical structures of the substrates and products. The transition states for chemical reactions are proposed to have lifetimes near 10-13 seconds, on the order of the time of a single bond vibration. No physical or spectroscopic method is available to directly observe the structure of the transition state for enzymatic reactions, yet transition state structure is central to understanding enzyme catalysis since enzymes work by lowering the activation energy of a chemical transformation.

It is now accepted that enzymes function to stabilize transition states lying between reactants and products, and that they would therefore be expected to bind strongly any inhibitor which closely resembles such a transition state. Substrates and products often participate in several enzyme reactions, whereas the transition state tends to be characteristic of one particular enzyme, so that such an inhibitor tends to be specific for that particular enzyme. The identification of numerous transition state inhibitors supports the transition state stabilization hypothesis for enzymatic catalysis.

Currently there is a large number of enzymes known to interact with transition state analogs, most of which have been designed with the intention of inhibiting the target enzyme. Examples include HIV-1 protease, racemases, β-lactamases, metalloproteinases, cyclooxygenases and many others.

Purine Nucleoside Phosphorylase

Purine nucleoside phosphorylase (PNP) is an enzyme involved in the catabolism and recycling of nucleosides and is a target for the development of novel therapeutic agents for T-cell apoptosis in leukemia and in autoimmune diseases. Inosine, guanosine, and 2’-deoxyguanosine are the major substrates for this enzyme (Figure 3 shows a representative PNP catalyzed reaction with inosine substrate).

Vern Schramm and colleagues at Albert Einstein College of Medicine have determined the transition state structure of PNP and used it to develop exquisitely tight binding transition state analogs to inhibit this enzyme. The PNP inhibitor Immucillin-H closely resembles the structure of the putative transition state (Figure 4) but is chemically stable due to substitution of the reactive C–N glycosidic bond with a C-C bond. The transition state of PNP bears an oxacarbenium ion in the sugar ring, and this is mimicked by the iminoribitol moiety of Immucillin-H.

Many transition state analogs exhibit properties of slow-binding inhibitors, where the inhibitor initially binds to form a weaker EI complex prior to a slow conformational change that leads to a very tight EI* complex.

and

K_i was determined by titrating Immucillin-H and measuring its effect on PNP initial rates v_o, and this value was 41 nM. K_i* was calculated from the same velocity measurements, but instead of using initial rates, second steady-state rates v_s were used, which corresponded to the steady-state inhibited rates following attainment of equilibrium for the slow-onset step when all of E has formed EI.

Stoichiometry and structural studies have revealed that one molecule of Immucillin-H binds to each subunit of the PNP trimer. However, inhibition assays indicated that occupancy of only one of the three is sufficient for complete inhibition of the trimer.

The design of Immucillin-H from an enzymatic transition-state analysis exemplifies a powerful approach for developing high-affinity enzyme inhibitors with pharmacological activity.