Tertiary structure, enzymes

However, proteases do not catalyze the hydrolysis of all kinds of proteins. Their action is stereo-selective Only proteins with a certain tertiary structure will be targeted. The reason is that some kind of orienting force is needed to place the amide group in the proper position for catalysis. The necessary contacts between an enzyme and its substrates (proteins) are created because the enzyme folds in such a way as to form a crevice into which the substrate fits the crevice also contains the catalytic groups. Therefore, proteins that do not fit into the crevice will not be hydrolyzed. This specificity preserves the integrity of other proteins such as hormones, and therefore the biological system continues to function normally, see also Enzymes Tertiary Structure. 

Noncompetitive inhibitors interact reversibly with enzymes to form an inactive species, effectively removing active enzyme and thus interfering with the rate of conversion of substrate to product. The inhibitor may interact with free enzyme, or with the enzyme-substrate complex. The key feature of noncompetitive inhibition that distinguishes it from competitive inhibition is that inhibition does not affect the apparent affinity of the enzyme for its substrate (i.e., the apparent Km). For example, a noncompetitive inhibitor may bind in a region remote from the active site to cause a reversible change in enzyme tertiary structure that completely prevents substrate binding and product formation. In this type of inhibition, the quantity of active enzyme appears to decrease as inhibitor concentration increases, so that the apparent Fmax for the reaction decreases. 

Either the solid support or the enzyme may be activated, but to limit disruption of the enzyme tertiary structure the functional groups of the support material are most often activated. The activation may occur prior to the coupling reaction (pre-activated supports), or a bi-functional linking reagent may be used to form the bond between... 

It is important to realize that developing methods for stabilizing the enzyme in its native form is also a major goal in this field (123). Successful progress along these lines have been obtained recently by the use of bifunctional reagents to cross-link the peptide chains within the enzyme tertiary structure. 

The shape of a large protein is influenced by many factors including of course Its primary and secondary structure The disulfide bond shown m Figure 27 18 links Cys 138 of carboxypeptidase A to Cys 161 and contributes to the tertiary structure Car boxypeptidase A contains a Zn " ion which is essential to the catalytic activity of the enzyme and its presence influences the tertiary structure The Zn ion lies near the cen ter of the enzyme where it is coordinated to the imidazole nitrogens of two histidine residues (His 69 His 196) and to the carboxylate side chain of Glu 72... 

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. 

Denaturation is accompanied by changes in both physical and biological properties. Solubility is drastically decreased, as occurs when egg white is cooked and the albumins unfold and coagulate. Most enzymes also lose all catalytic activity when denatured, since a precisely defined tertiary structure is required for their action. Although most denaturation is irreversible, some cases are known where spontaneous renaturation of an unfolded protein to its stable tertiary structure occurs. Renaturation is accompanied by a full recovery of biological activity. 

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. 

Enzymes frequently lose their activity and secondary and tertiary structural integrity immediately upon incorporation into w/o-MEs, particularly for systems containing ionic surfactants such as AOT [61-67], It is the author s belief that this occurs if INJ is not conducted quickly because of the exposure of biomolecules to macroscopic interfaces... 

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