Flexible enzyme hypothesis

Another hypothesis suggests that the binding of a substrate to an enzyme causes a strain or deformation of some of the bonds in the substrate molecule, which are subsequently broken. The effectiveness of this mechanism depends upon the strength of the binding force and does not necessarily involve any movement of the protein but suggests the idea of a flexible enzyme. 

To test the hypothesis that the conformational flexibility of the thermophilic enzyme is lower at room temperature than at higher temperatures, Kohen and Klinman measured, by FTIR, the time course of H/D exchange of protein N-H sites in deuterium oxide for the thermophilic alcohol dehydrogenase. Their measurements were made at the optimal host-organism temperature of 65 °C and at 25 °C, below the transition temperature. They also included yeast alcohol dehydrogenase at 25 °C, which is the optimal temperature for its own host organism. 

The conformation of membrane-bound enzymes is undoubtedly restricted by the membrane. However, the mechanism of action of these enzymes appears to be similar to that of soluble enzymes, so that the presence of clefts and conformational flexibility is to be expected. The mitochondrial coupling factor apparently contains both the ATP synthesizing enzyme and a proton channel conformational changes undoubtedly play a role in the function of this system. A large movement of polypeptide chains has been proposed in the functioning of this system (and for other membrane-bound enzymes), but no convincing experimental evidence is available to support such a hypothesis. 

Early enzymatic theory emphasized the importance of high complementarity between an enzyme s active site and the substrate. A closer match was thought to be better. This idea was formally described in Fischer s lock and key model. The role of an enzyme (E), however, is not simply to bind the substrate (S) and form an enzyme-substrate complex (ES) but instead to catalyze the conversion of a substrate to a product (P) (Scheme 4.2). Haldane, and later Pauling, stated that an enzyme binds the transition state (TS ) of the reaction. Koshland expanded this theory in his induced fit hypothesis.5 Koshland focused on the conformational flexibility of enzymes. As the substrate interacts with the active site, the conformation of the enzyme changes (E — E ). In turn, the enzyme pushes the substrate toward its reactive transition state (E TS ). As the product forms, it quickly diffuses out of the active site, and the enzyme assumes its original conformation. 

P450cam hydroxylates Ru-Cg-Ad when supplied with electrons via the natural NADH/putidaredoxin reductase/putd reduction relay.Ru-Cg-Ad hydroxylation occurs at only 1.6% the rate of camphor hydroxylation, and only 10% of the electrons supplied by NADH go to product formation. Presumably the rest are diverted to the formation of reduced oxygen species such as superoxide or hydrogen peroxide. The remarkable ability of P450cam to hydroxylate a molecule so structurally different from camphor supports the hypothesis that the structural flexibility inherent in the P450 fold allows these enzymes to hydroxylate structurally diverse substrates. 

This short presentation of integral solution techniques for biokinetic equations shows that integration of the function incorporating the hypothesis works only in the simplest cases. In addition, the integrated form lacks much of the flexibility desired for fitting data there is an immediate tendency to resort to descriptive polynomial functions. One also tries to use simplified forms of enzyme kinetics such as Monod kinetics, and this involves power law -type equations (cf. type 1 in Fig. 4.12 or Equ. 2.2a). 

An enzyme is typically a large protein molecule that contains one or more active sites where interactions with substrates take place. These sites are structurally compatible with specific substrate molecules, in much the same way as a key fits a particular lock. In fact, the notion of a rigid enzyme structure that binds only to molecules whose shape exactly matches that of the active site was the basis of an early theory of enzyme catalysis, the so-called lock-and-key theory developed by the German chemist Emil Fischer in 1894 (Figure 13.28). Fischer s hypothesis accounts for the specificity of enzymes, but it contradicts research evidence that a single enzyme binds to substrates of different sizes and shapes. Chemists now know that an enzyme molecule (or at least its active site) has a fair amount of structural flexibility and can modify its shape to accommodate more than one type of substrate. Figure 13.29 shows a molecular model of an enzyme in action. 

Several 3D protein structures of bacterial SiaTs have been determined in the presence of GMP-Neu5Ac as a ligand [60]. Based on these crystal structures, from which the active site organization and the catalytic mechanism have been elucidated, SiaT enzymes can be predicted to tolerate rather flexibly structural variations in the sialic acid part. This can be rationalized because only the nucleotide portion and the sialic acid substructure around the anomeric center become buried into the active site upon substrate binding and orientation toward the sialyl acceptor substrate, whereas much of the remainder of the siahc acid portion remains oriented toward the protein surface or even in contact with bulk solvent. This hypothesis has been verified by a number of preparative studies [33,47, 61). 

It is an old hypothesis that enzymes should be capable of efficiently catalyzing reactions with unfavorable entropies of activation by acting as entropy traps , i.e., that the binding energy of the enzyme is used to freeze out the rotational and translational degrees of freedom, necessary to form the activated complex. These effects seem to be smaller than previously thought since enzyme molecules are quite flexible. A qualitative examination of the entropic contribution to the rate acceleration of serine proteases indicated that this is small. ... 

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