Structure of enzymes

Smith, S.O., et al. Crystal versus solution structures of enzymes NMR spectroscopy of a crystalline serine protease. Science 244 961-964, 1989. 

Advances in the technology of x-ray diffraction have made it possible to achieve three-dimensional structures of enzymes together bound, in many cases, to their substrates. 

The structures of enzyme active sites, and other ligand binding pockets on enzymes, are ideally suited for high-affinity interactions with drug-like inhibitors. 

We have just discussed several common strategies that enzymes can use to stabilize the transition state of chemical reactions. These strategies are most often used in concert with one another to lead to optimal stabilization of the binary enzyme-transition state complex. What is most critical to our discussion is the fact that the structures of enzyme active sites have evolved to best stabilize the reaction transition state over other structural forms of the reactant and product molecules. That is, the active-site structure (in terms of shape and electronics) is most complementary to the structure of the substrate in its transition state, as opposed to its ground state structure. One would thus expect that enzyme active sites would bind substrate transition state species with much greater affinity than the ground state substrate molecule. This expectation is consistent with transition state theory as applied to enzymatic catalysis. 

Zuegg, J., Gruber, K., Gugganig, M. et al. (1999) Three-dimensional structures of enzyme-substrate complexes of the hydroxynitrile lyase from Hevea brasiliensis. Protein Science A Publication of the Protein Society, 8, 1990-2000. 

To close on a more positive note, we observe that the computed geometry of the enzyme-dienolate complex in the vicinity of the 3-carbonyl is insensitive to the assumed dielectric constant and is in close agreement with X-ray structures of enzyme-inhibitor complexes (see Table 4.11 and Fig. 4.15). It is really quite remarkable that 4 billion years of random walk by mother nature and a few hours of optimization with a quantum chemistry program such as Gaussian (starting with the correct functional groups) lead to the same structure for the active... 

Figure 1 Schematic representation of tomato ACS poiypeptide with marked a-heiicai and /3-strand secondary structure regions (according to PDB with entry code 1 iAX), residues criticai for cataiysis, and fragments of the poiypeptide representing the iarge and the smaii domain in the spatiai structure of enzyme. Open biocks denote a-heiicai regions and fiiied biocks, /3-strand regions. For detaiis see Sections 5.04.2.2.4 and 5.04.2.2.5.

Tbis explanation of enzyme action is belpful, but far from complete. For one thing, enzymes differ significantly in the ways that they interact with other compounds. Some enzymes bond and react with only specific compounds, while others bond and react with an array of compounds in a chemical family that have the same or similar functional groups. Some enzymes fit neatly into an opening in a substrate, while others actually change the shape of the substrate on which they operate. The fact that enzyme actions are so diverse simply conhrms that the chemical structures of enzymes and substrates differ signihcantly, and the chemical mechanisms by which they interact can be very complex indeed. In fact, the tools needed to understand the precise molecular shapes of enzymes and substrates have become available only recently. Once these shapes have become known, scientists are able to unravel the exact steps that take place when enzyme and substrate interact with each other. 

Because metal ions bind to and modify the reactivity and structure of enzymes and substrates, a wide spectrum of techniques has been developed to examine the nature of metal ions which serve as templates, redox-active cofactors, Lewis acids/bases, ion-complexing agents, etc. 

Figure 3.21 — (A) Integrated FET with two hydrogen ion-sensitive FET elements. (B) Structure of enzyme-modified FET sensor S plastic card FET enzyme-modified FET chip lUM, immobilized urease membrane. (C) Flowthrough cell Bl fixed sensor cell block B2 movable sensor cell block SC flowthrough cell EC electrical connector RP silicone rubber sheet AMP amplifier. (Reproduced from [151] with permission of Elsevier Science Publishers).

Cotton, F. A., Hazen, E. E., Jr., and Legg, M. J. (1979). Staphylococcal nuclease Proposed mechanism of action based on structure of enzyme-thymidine 3, 5 -bisphosphate-calcium ion complex at 1.5-A resolution. Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555. 

The dimensions of cavities in enzymes differ considerably, depending on their physiological function. In many cases the clefts are occupied by clusters of organized water molecules. Such clusters can be seen in certain X-ray structures of enzymes (e.g., the structure of carboxypeptidase A determined by Lipscomb). If the clefts are deep, as in horse liver alcohol dehydrogenase, a channel for removal of water is present (Branden). 

When 18-crown-6 was co-lyophilized with a-chymotrypsin, a 470-fold activation was seen over the free enzyme in the transesterification of APEE with 1-propanol in cyclohexane (Scheme 3.2) [96]. There was a low apparent specificity for the size and macrocyclic nature of the crown ether additives, suggesting that, during lyophilization, 18-crown-6 protects the overall native conformation and acts as a lyoprotectant. To examine this global effect, FTIR was used to examine the effect of crown ethers on the secondary structure of enzymes. In one study [98], subtilisin Carlsberg was shown to retain its secondary structure in 1,4-dioxane when lyophi-lized in a 1 1 ratio with 18-crown-6. In addition, examination of FTIR spectra from varying incubation temperatures indicated that an increase in crown ether content in the final enzyme preparation resulted in a decreased denaturation temperature in the solvent, indicating a more flexible protein structure. 

Huebner, G. Koenig, S. Koch, M.H.J. Hengstenberg, W. Influence of Phosphoenolpyruvate and Magnesium Ions on the Quaternary Structure of Enzyme I of the Phosphotransferase System from Gram-Positive Bacteria. Biochemistry, 34, 15700-15703 (1995)... 

T. Goody, R.S. Lavie, A. Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexes along the reaction coordinate. Structure, 8, 629-642 (2000)... 

In modem organisms, nucleic acids encode the genetic information that specifies the structure of enzymes, and enzymes catalyze the replication and repair of nucleic acids. The mutual dependence of these two classes of biomolecules brings up the perplexing question which came first, DNA or protein ... 

Impressed by the specificity of enzymatic action, biochemists early adopted a "lock-and-key" theory which stated that for a reaction to occur the substrate must fit into an active site precisely. Modem experiments have amply verified the idea. A vast amount of kinetic data on families of substrates and related competitive inhibitors support the idea and numerous X-ray structures of enzymes with bound inhibitors or with very slow substrates have given visual evidence of the reality of the lock-and-key concept. Directed mutation of genes of many enzymes of known three-dimensional structure has provided additional proof. 

Coenzyme and substrate analogs. The structures of enzyme NAD1 substrate complexes (Fig. 15-3) may be studied by X-ray crystallography under certain conditions or can be inferred from those of various stable enzyme-inhibitor complexes or from enzyme reconstituted with NAD+ that has been covalently... 

A relatively common feature of many problems involving molecular weight determination of biopolymers is that of association-dissociation equilibrium. Subunit structure of enzyme proteins is well recognized (1), and methods of dissociation of subunits to obtain monomer molecular weight are widely utilized (2). A previous paper described the application of an equilibrium gel partition method to the analysis of macromolecular association in a monomer-dimer case (3). The experimental parameters in a system utilizing the Sephadex series of gel filtra-... 

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