Enzymatics enzyme activity

FIGURE 2.16 pH versus enzymatic activity. The activity of enzymes is very sensitive to pH. The pH optimum of an enzyme is one of its most important characteristics. Pepsin is a protein-digesting enzyme active in the gastric fluid. Trypsin is also a proteolytic enzyme, but it acts in the more alkaline milieu of the small intestine. Lysozyme digests the cell walls of bacteria it is found in tears. 

FIGURE 14.12 The effect of temperature on enzyme activity. The relative activity of an enzymatic reaction as a fnncdon of tempera-tnre. The decrease in the activity above 50 C is dne to thermal denatnradon. 

If the inhibitor combines irreversibly with the enzyme—for example, by covalent attachment—the kinetic pattern seen is like that of noncompetitive inhibition, because the net effect is a loss of active enzyme. Usually, this type of inhibition can be distinguished from the noncompetitive, reversible inhibition case since the reaction of I with E (and/or ES) is not instantaneous. Instead, there is a time-dependent decrease in enzymatic activity as E + I El proceeds, and the rate of this inactivation can be followed. Also, unlike reversible inhibitions, dilution or dialysis of the enzyme inhibitor solution does not dissociate the El complex and restore enzyme activity. 

The first hint that two active-site carboxyl groups—one proto-nated and one ionized—might be involved in the catalytic activity of the aspartic proteases came from studies of the pH dependence of enzymatic activity. If an ionizable group in an enzyme active site is essential for activity, a plot of enzyme activity versus pH may look like one of the plots at right. 

No enzymatic side effects are observed and substrate concentrations up to 20% by weight can be used without affecting the enzyme activity. The biocatalyst is used in soluble form in a batch wise process, thus poorly soluble amino adds can be resolved without technical difficulties. Re-use of the biocatalyst is in prindple possible. 

The enzymatic transformation of natural products is by for file most attractive option. In this approach, it can be envisaged that sterols, which are relatively abundant, may be selectively modified to produce desired products. Hie diversity of enzyme activities, their reaction specificity, regiospecificity and stereospedfidty are all features which could contribute to carrying out the desired changes. This does not mean, however, that transformations using enzyme systems are simple. Nevertheless, biotransformations have become of vital importance in the production of steroids. 

As discussed in the early sections it seems that there are very few effective ways to stabilize the transition state and electrostatic energy appears to be the most effective one. In fact, it is quite likely that any enzymatic reaction which is characterized by a significant rate acceleration (a large AAgf +p) will involve a complimentarity between the electrostatic potential of the enzyme-active site and the change in charges during the reaction (Ref. 10). This point may be examined by the reader in any system he likes to study. 

Fungal chitin synthases are found as integral proteins of the plasma membrane and in chitosomes a divalent cation, Mg(II), is necessary for enzyme activity but neither primers nor a hpid intermediate are required. The substrate and free GlcNAc activate the allosteric enzyme. UDP, the byproduct of the enzymatic activity, is strongly inhibitory to chitin synthase however, it may be metabohzed readily to UMP by a diphosphatase. 

For bio-transformation processes, immobilised enzymes are often used because their activity persists over a longer period of time than that of free enzymes. The reduction of enzyme activity in enzymatic reactors is a consequence of energy dissipation by sparging and stirring, which is required for instance for oxygen transport or realisation of constant reaction conditions as regards temperature and pH. In the other hand low and high pH-values leads also to a decrease of enzyme activity and increase the stress sensitivity. 

The replacement of His 234 with Lys abolished the enzymatic activity, confirming the biochemical evidence obtained by Cooke et al. [13] and by Rexova-Benkova et aL [14] that a histidine readue is critical for the activity of the enzyme. Replacement of either Ser 237 or Ser 240 with Gly reduced the enzyme activity to 48% and 6% reqrectively, indicating that Ser residues are also inqrortant for the activity. 

Monitoring enzyme catalyzed reactions by voltammetry and amperometry is an extremely active area of bioelectrochemical interest. Whereas liquid chromatography provides selectivity, the use of enzymes to generate electroactive products provides specificity to electroanalytical techniques. In essence, enzymes are used as a derivatiz-ing agent to convert a nonelectroactive species into an electroactive species. Alternatively, electrochemistry has been used as a sensitive method to follow enzymatic reactions and to determine enzyme activity. Enzyme-linked immunoassays with electrochemical detection have been reported to provide even greater specificity and sensitivity than other enzyme linked electrochemical techniques. 

The simplest method of coupling enzymatic reactions to electrochemical detection is to monitor an off-line reaction using FIAEC or LCEC. The enzymatic reaction is carried out in a test tube under controlled conditions with aliquots being taken at timed intervals. These aliquots are then analyzed for the electroactive product and the enzyme activity in the sample calculated from the generated kinetic information. 

Lequea et al. used the activity of tyrosine apodecarboxylase to determine the concentration of the enzyme cofactor pyridoxal 5 -phosphate (vitamin B6). The inactive apoenzyme is converted to the active enzyme by pyridoxal 5 -phosphate. By keeping the cofactor the limiting reagent in the reaction by adding excess apoenzyme and substrate, the enzyme activity is a direct measure of cofactor concentration. The enzymatic reaction was followed by detecting tyramine formation by LCEC. The authors used this method to determine vitamin B6 concentrations in plasma samples. 

The diagnosis of PK deficiency depends on the determination of quantitative enzyme activity or qualitative abnormalities of the enzyme. In 1979, the International Committee for Standardization in Haematology (ICSH) established methods for the biochemical characterization of red blood cell PK variants (M22). Since the establishment of these methods, many PK-deficient cases have been characterized, including 13 cases of homozygous PK deficiency. Residual red blood cell PK activity is not usually associated with phenotypic severity,whereas enzymatic characteristics such as decreased substrate affinity, thermal instability, or impaired response to the allosteric activator fructose-1,6-diphosphate (F-1,6-DP) correspond to a more severe phenotype. 

All enzymatic reactions are initiated by formation of a binary encounter complex between the enzyme and its substrate molecule (or one of its substrate molecules in the case of multiple substrate reactions see Section 2.6 below). Formation of this encounter complex is almost always driven by noncovalent interactions between the enzyme active site and the substrate. Hence the reaction represents a reversible equilibrium that can be described by a pseudo-first-order association rate constant (kon) and a first-order dissociation rate constant (kM) (see Appendix 1 for a refresher on biochemical reaction kinetics) ... 

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. 

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... 

Enzymatic reactions are influenced by a variety of solution conditions that must be well controlled in HTS assays. Buffer components, pH, ionic strength, solvent polarity, viscosity, and temperature can all influence the initial velocity and the interactions of enzymes with substrate and inhibitor molecules. Space does not permit a comprehensive discussion of these factors, but a more detailed presentation can be found in the text by Copeland (2000). Here we simply make the recommendation that all of these solution conditions be optimized in the course of assay development. It is worth noting that there can be differences in optimal conditions for enzyme stability and enzyme activity. For example, the initial velocity may be greatest at 37°C and pH 5.0, but one may find that the enzyme denatures during the course of the assay time under these conditions. In situations like this one must experimentally determine the best compromise between reaction rate and protein stability. Again, a more detailed discussion of this issue, and methods for diagnosing enzyme denaturation during reaction can be found in Copeland (2000). 

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