Orientation of enzyme molecules

With infrared reflection absorption spectroscopy (IRRAS), it is possible to obtain information about the orientation of enzyme molecules adsorbed on flat metal surfaces (3,4). Electric dipole-transition moments oriented perpendicular to a flat metal surface show enhanced IR absorbance. IR bands due to vibrations of groups with transition moments oriented parallel to the surface are not observed. The IR-beam component which is polarized perpendicular to the plane of incidence (parallel to the surface) contains no information and can be eliminated by using a polarizer. 

Previously we determined that acid hydrolases were predominantly soluble within the lysosome matrix or were covalently linked to die vacuolar membrane (see, also, Koenig, 1962). While some data are available to suggest the nature of enzyme-membrane linkage, viz., through imidazole bonds (see previous section), the orientation of enzyme molecules in the vacuolar membrane is unknown. The action of enzymes at lipid/water interfaces is unclear, but Ryan and Mavrides (1960) have revealed, in vitro, an activation of -glucuronidase by varying the interface area between lipid solvents and buffered media. Such observations may reflect on the properties of enzymes at membrane interfaces. 

Fig. 3. Stereodiagrams of a-carbon atom chains of RNase-A and RNase-S from the studies by X-ray diffraction on the crystalline enzymes (a) RNase-A from the work of Kartha et al. (60). (b) RNase-S from the work of Wyckoff et al. (62). The orientation of the molecules has been made as similar as possible to simplify comparison. The figures are reproduced with the kind permission of Dickerson and Geis (63).

When two molecules approach one another to begin a chemical reaction, the probability of a successful encounter can depend critically on the three-dimensional shapes and the relative orientation of the molecules, as well as on their chemical identities. Shape is especially important in biological and biochemical reactions, in which molecules must fit precisely onto specific sites on membranes and templates drug and enzyme activity are important examples. Characterization of molecular shape is therefore an essential part of the study of molecular structure. 

Covalent immobilization strategies typically provide superior electrocatalytic characteristics, but the tethering can sometimes hinder protein conformation [66]. In addition, the functional groups on the enzyme that are used for tethering should not be essential to catalysis, or enzyme inactivation losses will occur. The ability to tailor covalent chemistry to specific regions of a protein, however, allows some control over protein orientation. A recent tendency in protein immobilization is the specific and rational orientation of protein molecules on a selected surface [67-69]. This strategy aims to create uniformly oriented protein immobilized in a manner that may optimize catalytic efficiency [70], enhance stability [68], or increase electron transfer efficiency [71,72]. 

Hein and Nieman, the substrates of the general type NH-CHR COR bind to three rather hydrophobic sites of the enzyme. We choose as leading example, for NTD-mapping of receptor sites, correlations of A =log (bimolecular rate constant) for hydrolysis of esters with NTD and also other parameters. We choose this area, because of the wealth of experimental data, because there is also other QSAR work for comparison, and because the orientation of the molecules in the receptor site of the enzyme will be well-defined, especially for molecules RCDNH-CHR -CQR of peptidic type, were RCONH will enter the p.-site... 

Figure 4.7 Two of the enzymatic activities involved in the biosynthesis of tryptophan in E. coli, phosphoribosyl anthranilate (PRA) isomerase and indoleglycerol phosphate (IGP) synthase, are performed by two separate domains in the polypeptide chain of a bifunctional enzyme. Both these domains are a/p-barrel structures, oriented such that their active sites are on opposite sides of the molecule. The two catalytic reactions are therefore independent of each other. The diagram shows the IGP-synthase domain (residues 48-254) with dark colors and the PRA-isomerase domain with light colors. The a helices are sequentially labeled a-h in both barrel domains. Residue 255 (arrow) is the first residue of the second domain. (Adapted from J.P. Priestle et al., Proc.

There is more to this story, however. Enzymes not only bring substrates and catalytic groups close together, they orient them in a manner suitable for catalysis as well. Comparison of the rates of reaction of the molecules shown... 

Enzymes work by bringing reactant molecules together, holding them, in the orientation necessary for reaction, and providing any necessary acidic or basic sites to catalyze specific steps. As an example, let s look at citrate synthase, an enzyme that catalyzes the aldol-like addition of acetyl CoA to oxaloacetate to give citrate. The reaction is the first step in the citric acid cycle, in which acetyl groups produced by degradation of food molecules are metabolized to yield C02 and H20. We ll look at the details of the citric acid cycle in Section 29.7. 

For molecules to react, they must come within bondforming distance of one another. The higher their concentration, the more frequently they will encounter one another and the greater will be the rate of their reaction. When an enzyme binds substrate molecules in its active site, it creates a region of high local substrate concentration. This environment also orients the substrate molecules spatially in a position ideal for them to interact, resulting in rate enhancements of at least a thousandfold. 

The high catalytic activity of enzymes has a number of sources. Every enzyme has a particular active site configured so as to secure intimate contact with the substrate molecule (a strictly defined mutual orientation in space, a coordination of the electronic states, etc.). This results in the formation of highly reactive substrate-enzyme complexes. The influence of tfie individual enzymes also rests on the fact that they act as electron shuttles between adjacent redox systems. In biological systems one often sees multienzyme systems for chains of consecutive steps. These systems are usually built into the membranes, which secures geometric proximity of any two neighboring active sites and transfer of the product of one step to the enzyme catalyzing the next step. 

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