Catalysis enzyme activity

Eor measurement of a substrate by a kinetic method, the substrate concentration should be rate-limiting and should not be much higher than the enzyme s K. On the other hand, when measuring enzyme activity, the enzyme concentration should be rate-limiting, and consequentiy high substrate concentrations are used (see Catalysis). 

Figure 5.9 Models of hexo-kinase in space-filling and wireframe formats, showing the cleft that contains the active site where substrate binding and reaction catalysis occur. At the bottom is an X-ray crystal structure of the enzyme active site, showing the positions of both glucose and ADP as well as a lysine amino acid that acts as a base to deprotonate glucose.

Hen egg-white lysozyme catalyzes the hydrolysis of various oligosaccharides, especially those of bacterial cell walls. The elucidation of the X-ray structure of this enzyme by David Phillips and co-workers (Ref. 1) provided the first glimpse of the structure of an enzyme-active site. The determination of the structure of this enzyme with trisaccharide competitive inhibitors and biochemical studies led to a detailed model for lysozyme and its hexa N-acetyl glucoseamine (hexa-NAG) substrate (Fig. 6.1). These studies identified the C-O bond between the D and E residues of the substrate as the bond which is being specifically cleaved by the enzyme and located the residues Glu 37 and Asp 52 as the major catalytic residues. The initial structural studies led to various proposals of how catalysis might take place. Here we consider these proposals and show how to examine their validity by computer modeling approaches. 

Hypothermia slows down enzyme catalysis of enzymes in plasma membranes or organelle membranes, as well as enzymes floating around in the cytosol. The primary reason enzyme activity is decreased is related to the decrease in molecular motion by lowering the temperature as expressed in the Arrhenius relationship (k = where k is the rate constant of the reaction, Ea the activation energy,... 

The mechanism for the lipase-catalyzed reaction of an acid derivative with a nucleophile (alcohol, amine, or thiol) is known as a serine hydrolase mechanism (Scheme 7.2). The active site of the enzyme is constituted by a catalytic triad (serine, aspartic, and histidine residues). The serine residue accepts the acyl group of the ester, leading to an acyl-enzyme activated intermediate. This acyl-enzyme intermediate reacts with the nucleophile, an amine or ammonia in this case, to yield the final amide product and leading to the free biocatalyst, which can enter again into the catalytic cycle. A histidine residue, activated by an aspartate side chain, is responsible for the proton transference necessary for the catalysis. Another important factor is that the oxyanion hole, formed by different residues, is able to stabilize the negatively charged oxygen present in both the transition state and the tetrahedral intermediate. 

Aminoacyl residues that participate in catalysis are highly conserved among all classes of a given enzyme activity. 

Enzymes accelerate reaction rates by lowering the activation barrier AGp. While they may undergo transient modification during the process of catalysis, enzymes emerge unchanged at the completion of the reaction. The presence of an enzyme therefore has no effect on AG for the overall reaction, which is a function solely of the initial and final states of the reactants. Equation (25) shows the relationship between the equilibrium constant for a reaction and the standard free energy change for that reaction ... 

Catalysis by flavoenzymes has been reviewed and various analogues of FAD have been prepared e.g. P -adenosine-P -riboflavin triphosphate and flavin-nicotinamide dinucleotide ) which show little enzymic activity. The kinetic constants of the interaction between nicotinamide-4-methyl-5-acetylimidazole dinucleotide (39) and lactic dehydrogenase suggest the presence of an anionic group near the adenine residue at the coenzyme binding site of the enzyme. ... 

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. 

The large molecular size and ambient operation of enzymes means that they are likely to be more suited to niche applications rather than to high-power devices, but there are important lessons to be leamt from biological catalysis that occurs in conditions under which conventional metal catalysts would fail. Development of synthetic catalysts inspired by the chemistry (although not necessarily the stmctures) of enzyme active sites may lead to future catalysts with new and improved properties. 

All soil metabolic proce.sses are driven by enzymes. The main sources of enzymes in soil are roots, animals, and microorganisms the last are considered to be the most important (49). Once enzymes are produced and excreted from microbial cells or from root cells, they face harsh conditions most may be rapidly decomposed by organisms (50), part may be adsorbed onto soil organomineral colloids and possibly protected against microbial degradation (51), and a minor portion may stand active in soil solution (52). The fraction of extracellular enzyme activity of soil, which is not denaturated and/or inactivated through interactions with soil fabric (51), is called naturally stabilized or immobilized. Moreover, it has been hypothesized that immobilized enzymes have a peculiar behavior, for they might not require cofactors for their catalysis. 

The active site of DHFR illustrates several features that are common to enzyme active sites. Some of the salient features of active site structure that relate to enzyme catalysis and ligand (e.g., inhibitor) interactions have been enumerated by Copeland (2000) ... 

It is worth noting here that inhibitors that interact with enzyme active site functionalities in ways that mimic the structure of covalent intermediates of catalysis can bind with very high affinity. This was seen in Chapter 1 with the example of statine-and hydroxyethylene-based inhibitors of aspartic proteases other examples of this inhibitor design strategy will be seen in subsequent chapters of this text. 

We have just discussed several common strategies that enzymes can use to stabilize the transition state of chemical reactions. These strategies are most often used in concert with one another to lead to optimal stabilization of the binary enzyme-transition state complex. What is most critical to our discussion is the fact that the structures of enzyme active sites have evolved to best stabilize the reaction transition state over other structural forms of the reactant and product molecules. That is, the active-site structure (in terms of shape and electronics) is most complementary to the structure of the substrate in its transition state, as opposed to its ground state structure. One would thus expect that enzyme active sites would bind substrate transition state species with much greater affinity than the ground state substrate molecule. This expectation is consistent with transition state theory as applied to enzymatic catalysis. 

The effects of solution pH on enzyme activity can be particularly informative in defining steps in catalysis that are most affected by interactions with inhibitors. Ionization of different groups on the enzyme can be critical in substrate binding (i.e.,... 

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... 

Because mechanism-based inactivation depends on enzyme catalysis, there cannot be more than one molecule of inactivator bound to the enzyme active site. Thus formation of the covalent E-A species cannot result in a stoichiometry of inactivator to enzyme of greater than 1 1. In the case of multimeric enzymes, however, it may not be necessary to covalently modify all of the enzyme active sites within the multi-mer in order to effect total inactivation of the enzyme. In this situation one may observe a stoichiometry of less that 1 1. Under no circumstances, however, can a mechanism-based inactivator display a stoichiometry of greater than 1 1 with the enzyme. 

Before discussing the mechanistic aspects of the PLCSc catalyzed hydrolysis of phospholipids, a brief survey of the manner in which metal ions in enzyme active sites participate in catalysis is warranted. 

As already stated, Fischer was deeply intrigued by the phenomenon of enzyme activity. He realized that the substances were proteins and this undoubtedly was why he next undertook the study of amino acids and peptides. He fully appreciated that the specificity of enzyme catalysis depended on the occurrence of a complementarity for interacting dissymmetric surfaces. In this regard, he wrote (3) ... 

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