Enzyme mechanisms active site studies

Quantum chemical methods aim to treat the fundamental quantum mechanics of electronic structure, and so can be used to model chemical reactions. Such quantum chemical methods are more flexible and more generally applicable than molecular mechanics methods, and so are often preferable and can be easier to apply. The major problem with electronic structure calculations on enzymes is presented by the very large computational resources required, which significantly limits the size of the system that can be treated. To overcome this problem, small models of enzyme active sites can be studied in isolation (and perhaps with an approximate model of solvation). Alternatively, a quantum chemical treatment of the enzyme active site can be combined with a molecular mechanics description of the protein and solvent environment the QM/MM approach. Both will be described below. 

The enzyme mechanism, however, remains elusive. Quantum mechanical models generally disfavor C6-protonation, but 02, 04, and C5-protonation mechanisms remain possibilities. Free energy computations also appear to indicate that C5-protonation is a feasible mechanism, as is direct decarboxylation without preprotonation O-protonation mechanisms have yet to be explored with these methods. Controversy remains, however, as to the roles of ground state destabilization, transition state stabilization, and dynamic effects. Because free energy models do take into account the entire enzyme active site, a comprehensive study of the relative energetics of pre-protonation and concerted protonation-decarboxylation at 02, 04, and C5 should be undertaken with such methods. In addition, quantum mechanical isotope effects are also likely to figure prominently in the ultimate identification of the operative ODCase mechanism. 

A relative of the kinases is adenylate cyclase, whose role in forming the allosteric effector 3, 5 -cyclic AMP (cAMP) was considered in Chapter 11. This enzyme catalyzes a displacement on P of ATP by the 3 -hydroxyl group of its ribose ring (see Eq. 11-8, step a). The structure of the active site is known. Studies with ATPotS suggest an in-line mechanism resembling that of ribonuclease (step a, Eq. 12-25). However, it is dependent, does not utilize the two-histidine mechanism of ribonuclease A, and involves an aspartate carboxylate as catalytic base. All isoforms of adenylate cyclase are activated by the a subunits of some G proteins (Chapter 11). The structures of Gg and of its complex with adenylate kinase have been determined. The Gg activator appears to serve as an allosteric effector. 

High-resolution n.m.r. combined with information from X-ray diffraction, is revealing much about the active sites and mechanisms of action of various enzymes, such as chymotrypsin (Gerig, 1968) and lysozyme (Cohen and Jardetzky, 1968), and the same benefits are expected for drugs and their receptors. Selective deuteration of the enzyme is a great help in these studies (Putter et al.y 1969). 

For many applications, especially studies on enzyme reaction mechanisms, we do not need to treat the entire system quantum mechanically. It is often sufficient to treat the center of interest (e.g., the active site and the reacting molecules) quantum mechanically. The rest of the molecule can be treated using classical molecular mechanics (MM see Section 7.2). The quantum mechanical technique can be ab-initio, DFT or semi-empirical. Many such techniques have been proposed and have been reviewed and classified by Thiel and co-workers [50] Two effects of the MM environment must be incorporated into the quantum mechanical system. 

Km for an enzymatic reaction are of significant interest in the study of cellular chemistry. From equation 13.19 we see that Vmax provides a means for determining the rate constant 2- For enzymes that follow the mechanism shown in reaction 13.15, 2 is equivalent to the enzyme s turnover number, kcat- The turnover number is the maximum number of substrate molecules converted to product by a single active site on the enzyme, per unit time. Thus, the turnover number provides a direct indication of the catalytic efficiency of an enzyme s active site. The Michaelis constant, Km, is significant because it provides an estimate of the substrate s intracellular concentration. 

A compound which is a good choice for an artificial electron relay is one which can reach the reduced FADH2 active site, undergo fast electron transfer, and then transport the electrons to the electrodes as rapidly as possible. Electron-transport rate studies have been done for an enzyme electrode for glucose (G) using interdigitated array electrodes (41). The following mechanism for redox reactions in osmium polymer—GOD biosensor films has... 

Uncovering of the three dimentional structure of catalytic groups at the active site of an enzyme allows to theorize the catalytic mechanism, and the theory accelerates the designing of model systems. Examples of such enzymes are zinc ion containing carboxypeptidase A 1-5) and carbonic anhydrase6-11. There are many other zinc enzymes with a variety of catalytic functions. For example, alcohol dehydrogenase is also a zinc enzyme and the subject of intensive model studies. However, the topics of this review will be confined to the model studies of the former hydrolytic metallo-enzymes. 

Spectroscopic studies have been instrumental in elucidating the catalytic mechanism of Ni-Fe hydrogenases. A great deal of controversy concerning this mechanism arises from the fact that, as the as the X-ray crystallographic analysis has shown, there are at least three potential redox-active species at the enzyme s active site the thiolate ligands (75) and the Fe (65) and Ni (9) ions. 

Inhibitors of the catalytic activities of enzymes provide both pharmacologic agents and research tools for study of the mechanism of enzyme action. Inhibitors can be classified based upon their site of action on the enzyme, on whether or not they chemically modify the enzyme, or on the kinetic parameters they influence. KineticaUy, we distinguish two classes of inhibitors based upon whether raising the substrate concentration does or does not overcome the inhibition. 

The reaction mechanism for glutamate racemase has been studied extensively. It has been proposed that the key for the racemization activity is that the two cysteine residues of the enzyme are located on both sides of the substrate bound to the active site. Thus, one cysteine residue abstracts the a-proton from the substrate, while the other detivers a proton from the opposite side of the intermediate enolate of the amino acid. In this way, the racemase catalyzes the racemization of glutamic acid via a so-called two-base mechanism (Fig. 15). 

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