Tertiary structure, of enzymes

The tertiary structure is the overall 3D shape of a protein. Structural proteins are quite ordered in shape, whereas other proteins such as enzymes and receptors fold up on themselves to form more complex structures. The tertiary structure of enzymes... 

Large proteins, which act as catalysts for biological reactions, are known as enzymes. The tertiary structure of enzymes usually produces 3-dimensional pockets known as the active sites. The size and shape of the active site is specific for only a certain type of substrate, which is selectively converted to the product by the enzyme. This is often compared with a key fitting a lock (the lock and key model). The catalytic activity of the enzyme is destroyed by denaturation, which is the breakdown of the tertiary structure (i.e. the protein unfolds). This can be caused by a change in temperature or pH. 

Since hydrogen bonds are relatively weak (1-5 kcal/mole) they can be easily dissociated, even by warming. The secondary and tertiary structure of enzymes (and other proteins) can be thermally unravelled, leading to destruction of catalytic activity. This protein denaturing process can also be brought about by chemical means such as with urea. 

Many chemicals can bind to enzymes and either eliminate or drastically reduce their catalytic ability. These chemicals, called enzyme inhibitors, have been used for hundreds of years. When she poisoned her victims with arsenic, Lucretia Borgia was unaware that it was binding to the thiol groups of cysteine amino acids in the proteins of her victims and thus interfering with the formation of disulfide bonds needed to stabilize the tertiary structure of enzymes. However, she was well aware of the deadly toxicity of heavy metal salts like arsenic and mercury. When you take penicillin for a bacterial infection, you are taking another enzyme inhibitor. Penicillin inhibits several enzymes that are involved in the synthesis of bacterial cell walls. 

In order to understand mechanisms of enzyme catalysis not only are the tertiary structures of enzymes of interest hut so too are the tertiary structures of enzyme-suhstrate complexes. There was, however, a problem As incredibly efficient catalysts, enzymes turn over substrate molecules in fractions of a second, while (40 years ago) collection of crystallographic data took days. The answer to the problem was to study complexes of exceedingly sluggish substrates, unreactive model substrates, as well as strongly bound inhibitors that compete for the active site. 

The basic kinetic properties of this allosteric enzyme are clearly explained by combining Monod s theory and these structural results. The tetrameric enzyme exists in equilibrium between a catalytically active R state and an inactive T state. There is a difference in the tertiary structure of the subunits in these two states, which is closely linked to a difference in the quaternary structure of the molecule. The substrate F6P binds preferentially to the R state, thereby shifting the equilibrium to that state. Since the mechanism is concerted, binding of one F6P to the first subunit provides an additional three subunits in the R state, hence the cooperativity of F6P binding and catalysis. ATP binds to both states, so there is no shift in the equilibrium and hence there is no cooperativity of ATP binding. The inhibitor PEP preferentially binds to the effector binding site of molecules in the T state and as a result the equilibrium is shifted to the inactive state. By contrast the activator ADP preferentially binds to the effector site of molecules in the R state and as a result shifts the equilibrium to the R state with its four available, catalytically competent, active sites per molecule. 

The class A enzymes have Mx values around 30,000. Their substrate specificities are quite variable and a large number of enzymes have emerged in response to the selective pressure exerted by the sometimes abusive utilization of antibiotics. Some of these new enzymes are variants of previously known enzymes, with only a limited number of mutations (1 4) but a significantly broadened substrate spectrum while others exhibit significantly different sequences. The first category is exemplified by the numerous TEM variants whose activity can be extended to third and fourth generation cephalosporins and the second by the NMCA and SME enzymes which, in contrast to all other SXXK (3-lactamases, hydrolyse carbapenems with high efficiency. Despite these specificity differences, the tertiary structures of all class A (3-lactamases are nearly superimposable. 

Figure 5-6. Examples of tertiary structure of proteins. Top The enzyme triose phosphate isomerase. Note the elegant and symmetrical arrangement of alternating p sheets and a helices. (Courtesy of J Richardson.) Bottom Two-domain structure of the subunit of a homodimeric enzyme, a bacterial class II HMG-CoA reductase. As indicated by the numbered residues, the single polypeptide begins in the large domain, enters the small domain, and ends in the large domain. (Courtesy ofC Lawrence, V Rod well, and C Stauffacher, Purdue University.)...

The CD spectrum of the C188S mutant is essentially the same as that of the wild-type enzyme, which reflects that the tertiary structure of this mutant changed little compared to that of the wild-type enzyme. Calculation of the content of secondary structure of the mutant enzyme based on J-600S Secondary Structure Estimation system (JASCO) also showed that there is no significant change in the secondary structure of the mutant. The fact that the k value of this mutant is extremely small despite little change in conformation clearly indicates that Cysl88 is located in the active site. 

However, this is not so easy without the tertiary structure of the enzyme. The possible clues are the homology search with functionally resembling enzymes and computer simulation of the tert-structure of the enzyme. The characteristic features of AMDase are (i) the reaction proceeds via an enolate-type transition state, (ii) the cysteine residue plays an essential role and (iii) the reaction involves an inversion of configuration on the a-carbon of the carboxyl group. 

Figure 2.10 Secondary and tertiary structure of the copper enzyme azurin visualized using Wavefunction, Inc. Spartan 02 for Windows from PDB data deposited as 1JOI. See text for visualization details. Printed with permission of Wavefunction, Inc., Irvine, CA. (See color plate.)...

Cook, W. J., Jeffrey, L. C., Xu, Y., and Chau, V. Tertiary structures of class 1 ubiquitin-conjugating enzymes are highly conserved crystal structure of yeast Ubc4. Biochemistry 1993, 32, 13809-17. 

Figure 2.10 Secondary and tertiary structure of the enzyme lysozyme, PDB 2C80. Visualized using Cambridge Soft Chem3D Ultra 10.0 with notations in ChemDraw Ultra 10.0. ChemDraw Ultra, version 10.0. (Printed with permission of CambridgeSoft...

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