Enzyme rate enhancements

Enzymes occur in every living cell and are the basic elements in the execution and control of metabolic processes. They are very sophisticated catalysts. In addition to bringing about spectacular rate enhancements, enzymes... 

Catalyst This is a generic name for a molecule that when added to a reaction causes rate enhancement. Enzymes are natural catalysts. There are also catalysts that have been invented by chemists for example, Fritz Haber used metal-based catalysts like osmium (Os) and iron (Fe). 

Most reactions in cells are carried out by enzymes [1], In many instances the rates of enzyme-catalysed reactions are enhanced by a factor of a million. A significantly large fraction of all known enzymes are proteins which are made from twenty naturally occurring amino acids. The amino acids are linked by peptide bonds to fonn polypeptide chains. The primary sequence of a protein specifies the linear order in which the amino acids are linked. To carry out the catalytic activity the linear sequence has to fold to a well defined tliree-dimensional (3D) stmcture. In cells only a relatively small fraction of proteins require assistance from chaperones (helper proteins) [2]. Even in the complicated cellular environment most proteins fold spontaneously upon synthesis. The detennination of the 3D folded stmcture from the one-dimensional primary sequence is the most popular protein folding problem. 

In contrast to the situation in the absence of catalytically active Lewis acids, micelles of Cu(DS)2 induce rate enhancements up to a factor 1.8710 compared to the uncatalysed reaction in acetonitrile. These enzyme-like accelerations result from a very efficient complexation of the dienophile to the catalytically active copper ions, both species being concentrated at the micellar surface. Moreover, the higher affinity of 5.2 for Cu(DS)2 compared to SDS and CTAB (Psj = 96 versus 61 and 68, respectively) will diminish the inhibitory effect due to spatial separation of 5.1 and 5.2 as observed for SDS and CTAB. 

In contrast to SDS, CTAB and C12E7, CufDSjz micelles catalyse the Diels-Alder reaction between 1 and 2 with enzyme-like efficiency, leading to rate enhancements up to 1.8-10 compared to the reaction in acetonitrile. This results primarily from the essentially complete complexation off to the copper ions at the micellar surface. Comparison of the partition coefficients of 2 over the water phase and the micellar pseudophase, as derived from kinetic analysis using the pseudophase model, reveals a higher affinity of 2 for Cu(DS)2 than for SDS and CTAB. The inhibitory effect resulting from spatial separation of la-g and 2 is likely to be at least less pronoimced for Cu(DS)2 than for the other surfactants. 

Mutations in the specificity pocket of trypsin, designed to change the substrate preference of the enzyme, also have drastic effects on the catalytic rate. These mutants demonstrate that the substrate specificity of an enzyme and its catalytic rate enhancement are tightly linked to each other because both are affected by the difference in binding strength between the transition state of the substrate and its normal state. 

Enigmas abound in the world of enzyme catalysis. One of these surrounds the discussion of how the rate enhancement by an enzyme can be best expressed. Notice that the nncatalyzed conversion of a substrate S to a product P is usually a simple first-order process, described by a first-order rate constant... 

If the rate enhancement effected by the enzyme is defined as rate enhancement = v /... 

Viewed in this way, the best definition of rate enhancement depends upon the relationship between enzyme and substrate concentrations and the enzyme s kinetic parameters. 

Figure 26.8 Energy diagrams for uncatalyzed (red) and enzyme-catalyzed (blue) processes. The enzyme makes available an alternative, lower-energy pathway. Rate enhancement is due to the ability of the enzyme to bind to the transition state for product formation, thereby lowering its energy.

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. 

Condensation of CO2, ammonia, and ATP to form carbamoyl phosphate is catalyzed by mitochondrial carbamoyl phosphate synthase I (reaction 1, Figure 29-9). A cytosolic form of this enzyme, carbamoyl phosphate synthase II, uses glutamine rather than ammonia as the nitrogen donor and functions in pyrimidine biosynthesis (see Chapter 34). Carbamoyl phosphate synthase I, the rate-hmiting enzyme of the urea cycle, is active only in the presence of its allosteric activator JV-acetylglutamate, which enhances the affinity of the synthase for ATP. Formation of carbamoyl phosphate requires 2 mol of ATP, one of which serves as a phosphate donor. Conversion of the second ATP to AMP and pyrophosphate, coupled to the hydrolysis of pyrophosphate to orthophosphate, provides the driving... 

At the present time, "interest in reversed micelles is intense for several reasons. The rates of several types of reactions in apolar solvents are strongly enhanced by certain amphiphiles, and this "micellar catalysis" has been regarded as a model for enzyme activity (. Aside from such "biomimetic" features, rate enhancement by these surfactants may be important for applications in synthetic chemistry. Lastly, the aqueous "pools" solubilized within reversed micelles may be spectrally probed to provide structural information on the otherwise elusive state of water in small clusters. 

Miller and Wolfenden, 2002). This latter ratio is the inverse of the rate enhancement achieved by the enzyme. In other words, the enzyme active site will have greater affinity for the transition state structure than for the ground state substrate structure, by an amount equivalent to the fold rate enhancement of the enzyme (rearranging, we can calculate KJX = Ksik Jk, )). Table 2.2 provides some examples of enzymatic rate enhancements and the calculated values of the dissociation constant for the /A binary complex (Wolfenden, 1999). 

In this chapter we have seen that enzymatic catalysis is initiated by the reversible interactions of a substrate molecule with the active site of the enzyme to form a non-covalent binary complex. The chemical transformation of the substrate to the product molecule occurs within the context of the enzyme active site subsequent to initial complex formation. We saw that the enormous rate enhancements for enzyme-catalyzed reactions are the result of specific mechanisms that enzymes use to achieve large reductions in the energy of activation associated with attainment of the reaction transition state structure. Stabilization of the reaction transition state in the context of the enzymatic reaction is the key contributor to both enzymatic rate enhancement and substrate specificity. We described several chemical strategies by which enzymes achieve this transition state stabilization. We also saw in this chapter that enzyme reactions are most commonly studied by following the kinetics of these reactions under steady state conditions. We defined three kinetic constants—kai KM, and kcJKM—that can be used to define the efficiency of enzymatic catalysis, and each reports on different portions of the enzymatic reaction pathway. Perturbations... 

Biochemical catalysts. Some reactions can catalyzed be by enzymes. The attraction in using enzymes rather than microorganisms is an enormous rate enhancement that can be obtained in the absence of the microorganisms. This is restricted to situations when the enzyme can be isolated and is also stable. In addition, the chemical reaction does not have to cater for the special requirements of living cells. 

A key question in the action of enzymes is the understanding of the mechanisms by which they attain their catalytic rate enhancement relative to the uncatalyzed reactions. Some enzymes have been shown to produce rate accelerations as large as 1019 [1], The theoretical determination of the reaction mechanisms by which enzymes carry out the chemical reactions has been an area of great interests and intense development in recent years [2-11], A common approach for the modeling of enzyme systems is the QM/MM method proposed by Warshel and Levitt [12], In this method the enzyme is divided into two parts. One part includes the atoms or molecules that participate in the chemical process, which are treated by quantum mechanical calculations. The other contains the rest of the enzyme and the solvent, generally thousands of atoms, which is treated by molecular mechanics methods. 

Such large rate enhancements naturally excite our curiosity how does Nature achieve such incredible feats Knowing something about the design features of enzymes, how can we improve other catalysts, to achieve reactions... 

Further research in improving the BDS activity of the biocatalysts was targeted towards the search of co-catalysts and co-factors to enhance overall desulfurization rates as well as promoters to enhance enzyme expression. This research resulted in identification of NADH and FMNH2 as co-factors essential for electron transfer and related oxidoreductase enzymes as co-catalysts as described in detail below. Additionally, other bacterial strains were also investigated as hosts and are reported below. 

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